Root Genomics

Root Genomics
.
Antonio Costa de Oliveira l Rajeev K. Varshney
Editors
Root Genomics
Editors
Professor Antonio Costa de Oliveira
Plant Genomics and Breeding Center
Federal University of Pelotas
Campus Universitario s/n, FAEM 3 andar
Pelotas-RS 96001-970,
Brazil
acostol@terra.com.br
Rajeev K. Varshney
Principal Scientist - ICRISAT;
Theme Leader, CGIAR Generation Challenge Programme;
Adjunct Professor, The University of Western Australia
Centre of Excellence in Genomics (CEG)
ICRISAT
Patancheru 502 324, A.P.,
India
r.k.varshney@cgiar.org
ISBN 978-3-540-85545-3 e-ISBN 978-3-540-85546-0

DOI 10.1007/978-3-540-85546-0
Springer Heidelberg Dordrecht London New York
# Springer-Verlag Berlin Heidelberg 2011

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Cover illustration: Roots of White Lupin (Lupinus albus). The photo has been taken from Drs. Bruna
Bucciarelli and Carroll Vance, U.S. Department of Agriculture, Agriculture Research Service, St. Paul,
Minnesota 55108, USA and the editors are grateful for the same.
Cover design: deblik Berlin, Germany
Printed on acid-free paper
Springer is part of Springer Science+Business Media (www.springer.com)

Foreword
Root Biology: An Inconvenient Truth
The truth is that roots usually are as extensively underground as the aerial portions
are above the ground. Crop plants would not live without roots. Roots absorb water
and nutrients and anchor the plant in the soil. So why do not we know more about
roots? It is likely due to the inconvenience of phenotyping root characteristics – and
many of today’s phenotyping methods are destructive. While we recognize the
essentiality of roots and their relation to plant performance, the scientific commu-
nity has not placed a sufficiently high priority on their analysis to make the needed
major advances. Many of the factors that affect root health can result in a 50% yield
loss when deficient. Given that the predicted human population increase is 50% by
2050, the improvement of root health in crop plants could play a major role in
meeting the world’s need for increased food.
The study of root biology involves extensive plant–soil–water interactions that
are complicated by the microorganisms and insects in the rhizosphere that can alter
root development. Each of the possible interactions has feedback effects in the
plant; many effects are long-range effects within the plant. The soil environment
relates to nutrient availability and uptake, which reflects the condition of the soil
including acidity. Even alternation of dry and flooded conditions changes various
ion states, which can change with the duration of flooding. Many climate change
scenarios predict water shortages, making the understanding of root biology even
more important in the future.
Much of today’s phenotyping of roots is based on root architecture, such as root
length, root diameter, root proliferation, root biomass, root mass density at different
soil depths, diameter, and distribution of meta-xylem vessels, and root-to-shoot
ratios. Early maturity, early shoot-growth vigor, and depth and rapidity of water
absorption also are often assessed among other factors. New nondestructive
approaches need to be encouraged such as X-ray imaging, light transmission
imaging, and time-lapse recordings of root growth.
v
vi Foreword
This book clearly documents that many new genetic/genomic technologies are
rapidly being applied to the study of roots, including high-throughput genome
sequencing, TILLING, use of molecular markers such as SSRs, DArTs, and SNPs
for introgression of favorable genes, QTL analyses, marker assisted breeding, gene
discovery, comparative mapping, transcription factor identification, transcriptional
profiling, posttranscriptional events regulating microRNAs, and proteome profiling
with complete roots. Some genetic approaches are constrained – such as genome-
wide selection and gene cloning – by the difficulty in phenotyping.
Plants coordinate root growth with the soil environment. Many factors can
inhibit root growth. In this book, aluminum, iron, and salt toxicity are extensively
reviewed, providing a great deal of useful information. The root system is the
primary site of interaction with the soil environment, which includes exudates of
organic compounds from the plants and the microbes. Some of these exudates are
known to represent signals that regulate microbe behaviors and even germination
of seeds.
As illustrated in this book, it is amazing what we know about roots and their
importance, but equally amazing is what we do not know – and we know even less
about the complicated interactions and feedback mechanisms. The work reviewed
in this book also shows the value of using model species such as Arabidopsis; e.g.,
22 genes have been reported in Arabidopsis on lateral root development, 19 genes
on primary root development, and 8 genes on root-hair formation.
One of the goals of this book was to show how root research relates to sustain-
able crop productivity. The chapters taken together represent an extensive review of
the topic focusing primarily on highly productive crops under rainfed conditions.
Crops are mostly rainfed in the most populated areas of the world; this suggests that
it is imperative that root biology be a major research emphasis in the coming years –
but will that be the case? Will the “inconvenient truth” be recognized?
Ronald L. Phillips
Regents Professor Emeritus
Department of Agronomy and Plant Genetics
Microbial and Plant Genomics Institute
University of Minnesota
St. Paul, MN 55108, USA
Preface
With the emerging recognition that agriculture needs to approach sustainability,

the plant–soil–water interactions become of paramount importance in crop systems.
In this scenario, roots arise from a minor to a major role in the understanding of
plant growth and development. Novel technologies allow us to scan genomes in
the fastest way ever, and there is not a day without further developments leading
to cheaper and more precise genotyping techniques. However, the complexity of
underground metabolism and the responses of root systems to a variety of stresses
call for improvements in phenotyping as well as genotyping techniques.
The idea of organizing a book on Root Genomics dates as back as early 1990s in
the graduate benches of Purdue University. The fascination with a system so
important for the plant but yet so unknown served as both an incentive and a
challenge to pursue this line of research. In 2002, an important opening for root
biology occurred when the late Dr. Mike Gale, FRS, agreed to include a workshop
in Root Genomics at the Plant and Animal Genome Meetings, held yearly at San
Diego, CA. Since 2003, this workshop has generated fruitful discussions and
created new paths for root research. Many speakers from different countries shared
their experience in root genomics, regardless if they were working with model or
crop species. One of the speakers, Rajeev Varshney, was very impressive in his
enthusiasm and determination to target important aspects of drought stress. Sharing
the same enthusiasm for studying roots and stress responses was crucial to put the
idea of this book forward. Many of the authors have presented their work in the
Root Genomics Workshop, but all were chosen by their significant contributions to
agricultural and plant sciences and their common efforts for a better world. We are
grateful to all the authors who not only provided a timely review of the published
research work in their area of expertise but also shared their unpublished results to
offer an updated view. We also appreciate their cooperation in meeting the dead-
lines, revising the manuscripts and in checking the galley-proofs.
We are thankful to Dr Jeff L. Bennetzen, who as a brilliant geneticist was a
great role model and a friend (ACO) that has indirectly inspired this line of research.
We thank Dr. Ronald Phillips, a major pioneer in the field of plant genetics and
vii
viii Preface
genomics and the father of many ideas that influenced modern plant sciences, for
writing the foreword.
Both of us also recognize that the editorial work for this book took away pre-
cious time that we should have spent with our respective families. ACO acknowl-
edges the efforts of his parents, Glauco and Izabel, for providing an atmosphere of
learning and investigative thought during his young years, his wife Carla for her
continuous encouragement, patience, and friendship, and his children Victoria
(Vickie) and Eduardo (Dudu). Similarly, RKV acknowledges the help and support
of his wife Monika and his children Prakhar (Kutkut) and Preksha (Nanu) who
allowed their time to be taken away to fulfill RKV’s editorial responsibilities in
addition to research, managerial, and other administrative duties at ICRISAT and
Generation Challenge Programme (GCP).
Pelotas-RS, Brazil Antonio Costa de Oliveira

Patancheru, A.P., India Rajeev K. Varshney
Contents
1 Introduction to Root Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Antonio Costa de Oliveira and Rajeev K. Varshney
2 EST-Based Approach for Dissecting Root Architecture

in Barley Using Mutant Traits of Other Species . . . . . . . . . . . . . . . . . . . . . . 11
Beata Orman, Aleksander Ligeza, Iwona Szarejko,
and Miroslaw Maluszynski
3 Genomics of Root–Microbe Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Ulrike Mathesius and Giel E. van Noorden
4 Plant Genetics for Study of the Roles of Root Exudates

and Microbes in the Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Aparna Deshpande, Ana Clara Pontaroli, Srinivasa R. Chaluvadi,
Fang Lu, and Jeffrey L. Bennetzen
5 Impact of the Environment on Root Architecture

in Dicotyledoneous Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Véronique Gruber, Ons Zahaf, Anouck Diet, Axel de Zélicourt,
Laura de Lorenzo, and Martin Crespi
6 Mechanisms of Aluminum Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Owen A. Hoekenga and Jurandir V. Magalhaes
7 Root Responses to Major Abiotic Stresses in Flooded Soils . . . . . . . . . 155

Rogerio O. Sousa and Antonio Costa de Oliveira
8 Genomics of Root Architecture and Functions in Maize . . . . . . . . . . . . 179

Roberto Tuberosa, Silvio Salvi, Silvia Giuliani, Maria Corinna
Sanguineti, Elisabetta Frascaroli, Sergio Conti, and Pierangelo Landi
ix
x Contents
9 Phenotyping for Root Traits and Their Improvement Through

Biotechnological Approaches for Sustaining Crop Productivity . . . . 205
M.S. Sheshshayee, Ehab Abou-Kheir, Rohini Sreevathsa,
Namita Srivastava, B. Mohanraju, Karaba N. Nataraja,
T.G. Prasad, and M. Udayakumar
10 Genomics and Physiological Approaches for Root Trait Breeding

to Improve Drought Tolerance in Chickpea (Cicer arietinum L.) . . . 233
Rajeev K. Varshney, Lekha Pazhamala, Junichi Kashiwagi,
Pooran M. Gaur, L. Krishnamurthy, and Dave Hoisington
11 Molecular Breeding of Cereals for Aluminum Resistance . . . . . . . . . . 251

Harsh Raman and Perry Gustafson
12 Molecular Breeding of Rice for Problem Soils . . . . . . . . . . . . . . . . . . . . . . . 289

Abdelbagi M. Ismail and Michael J. Thomson
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Contributors
Ehab Abou-Kheir Department of Crop Physiology, University of Agricultural

Sciences, GKVK Campus, Bangalore 560065, India
Jeffrey L. Bennetzen Department of Biological Sciences, Purdue University,

West Lafayette, IN 47906, USA; Department of Genetics, University of Georgia,
Athens, GA 30602, USA; Department of Biological Sciences, Michigan Techno-
logical University, Houghton, MI 49931, USA
Srinivasa R. Chaluvadi Department of Genetics, University of Georgia, Athens,

GA 30602, USA
Sergio Conti Department of Agroenvironmental Sciences and Technology, Viale

Fanin 44, 40127, Bologna, Italy
Antonio Costa de Oliveira Plant Genomics and Breeding Center, Eliseu Maciel
School of Agronomy, Federal University of Pelotas, Campus UFPel, Capão do
Leão RS-96001-970, Brazil
Martin Crespi Institut des Sciences du Végétal, C.N.R.S, 91198 Gif-sur-Yvette,

France
Laura de Lorenzo Institut des Sciences du Végétal, C.N.R.S, 91198 Gif-sur-

Yvette, France
Axel de Zélicourt Institut des Sciences du Végétal, C.N.R.S, 91198 Gif-sur-

Yvette, France, Université Paris Diderot Paris 7, Les Grands Moulins, 16 rue
Marguerite Duras, 75 205 Paris Cedex 13, France
Aparna Deshpande Department of Biological Sciences, Purdue University, West

Lafayette, IN 47906, USA; Department of Biological Sciences, Michigan Techno-
logical University, Houghton, MI 49931, USA
xi
xii Contributors
Anouck Diet Institut des Sciences du Végétal, C.N.R.S, 91198 Gif-sur-Yvette,

France; Université Paris Diderot Paris 7, Les Grands Moulins, 16 rue Marguerite
Duras, 75 205 Paris Cedex 13, France
Elisabetta Frascaroli Department of Agroenvironmental Sciences and

Technology, Viale Fanin 44, 40127 Bologna, Italy
Pooran M. Gaur International Crops Research Institute for the Semi-Arid Tropics
(ICRISAT), Patancheru 502324, A.P., India
Silvia Giuliani Department of Agroenvironmental Sciences and Technology,

Viale Fanin 44, 40127 Bologna, Italy
Véronique Gruber Institut des Sciences du Végétal, C.N.R.S, 91198 Gif-sur-

Yvette, France; Université Paris Diderot Paris 7, Les Grands Moulins, 16 rue
Marguerite Duras, 75 205 Paris Cedex 13, France
Perry Gustafson USDA-ARS, University of Missouri, 206 Curtis Hall, Columbia,

MO 65211, USA
Owen A. Hoekenga US Department of Agriculture, Robert W. Holley Center for

Agriculture and Health, Agricultural Research Service, Ithaca, NY 14853, USA
Dave Hoisington International Crops Research Institute for the Semi-Arid Tropics
(ICRISAT), Patancheru 502324, A.P., India
Abdelbagi M. Ismail International Rice Research Institute (IRRI), DAPO 7777,

Metro Manila, Philippines
Junichi Kashiwagi Graduate School of Agriculture, Hokkaido University, Kita 9

Nishi 9, Kita-ku, Sapporo 060-8589, Japan
L. Krishnamurthy International Crops Research Institute for the Semi-Arid

Tropics (ICRISAT), Patancheru 502324, A.P., India
Pierangelo Landi Department of Agroenvironmental Sciences and Technology,

Aleksander Ligeza Department of Genetics, Silesian University, Katowice,

Poland
Fang Lu Department of Genetics, University of Georgia, Athens, GA 30602, USA
Jurandir V. Magalhaes Embrapa Maize and Sorghum, Rod. MG 424 Km 65,

35701-970 Sete Lagoas, Minas Gerais, Brazil
Contributors xiii
Miroslaw Maluszynski Department of Genetics, Silesian University, Katowice,

Poland
Ulrike Mathesius Australian Research Council Centre of Excellence for Integra-

tive Legume Research, School of Biochemistry and Molecular Biology, Australian
National University, Linnaeus Way, Canberra, ACT 0200, Australia
B. Mohanraju Department of Crop Physiology, University of Agricultural

Karaba N. Nataraja Department of Crop Physiology, University of Agricultural

Beata Orman Department of Genetics, Silesian University, Katowice, Poland
Lekha Pazhamala International Crops Research Institute for the Semi-Arid Tro-
pics (ICRISAT), Patancheru 502324, A.P., India
Ana Clara Pontaroli Department of Genetics, University of Georgia, Athens, GA,

30602, USA; Estación Experimental Agropecuaria Balcarce, Instituto Nacional de
Tecnologı́a Agropecuaria (INTA) – Consejo Nacional de Investigaciones Cientı́fi-
cas y Técnicas (CONICET), CC 276 (7620), Balcarce, Argentina
T.G. Prasad Department of Crop Physiology, University of Agricultural Sciences,

GKVK Campus, Bangalore 560065, India
Harsh Raman NSW Department of Industry and Investment, Wagga Wagga

Agricultural Institute, Wagga Wagga, NSW 2650, Australia
Silvio Salvi Department of Agroenvironmental Sciences and Technology, Viale

Fanin 44, 40127 Bologna, Italy
Maria Corinna Sanguineti Department of Agroenvironmental Sciences and

Technology, Viale Fanin 44, 40127 Bologna, Italy
M.S. Sheshshayee Department of Crop Physiology, University of Agricultural

Rogerio O. Sousa Department of soils, Eliseu Maciel School of Agronomy,

Federal University of Pelotas, Campus UFPel, Capão do Leão RS-96001-970,
Brazil
Rohini Sreevathsa Department of Crop Physiology, University of Agricultural

xiv Contributors
Namita Srivastava Department of Crop Physiology, University of Agricultural

Iwona Szarejko Department of Genetics, Silesian University, Katowice, Poland
Michael J. Thomson International Rice Research Institute (IRRI), DAPO 7777,

Metro Manila, Philippines
Roberto Tuberosa Department of Agroenvironmental Sciences and Technology,

M. Udayakumar Department of Crop Physiology, University of Agricultural

Giel E. van Noorden Australian Research Council Centre of Excellence for

Integrative Legume Research, School of Biochemistry and Molecular Biology,
Australian National University, Linnaeus Way, Canberra, ACT 0200, Australia
Rajeev K. Varshney Centre of Excellence in Genomics (CEG), International

Crops Research Institute for the Semi-Arid Tropics, Patancheru 502 324, A.P.,
India; Theme - Comparative and Applied Genomics, Generation Challenge
Programme, c/o CIMMYT, Int APDO, Postal 6-641, 06600 Mexico, DF, Mexico;
School of Plant Biology (M084), The University of Western Australia, 35 Stirling
Highway, Crawley, WA 6009, Australia
Ons Zahaf Institut des Sciences du Végétal, C.N.R.S, 91198 Gif-sur-Yvette,

France
Chapter 1
Introduction to Root Genomics
Antonio Costa de Oliveira and Rajeev K. Varshney
Contents
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Root Genomics: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.1 Root Growth and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.2 Biotic Stress Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2.3 Abiotic Stress Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2.4 QTL Analysis and Molecular Breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3 About the Book . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.4 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.1 Introduction
The twenty first century has been marked by climate awareness and an overall
increase in conscience towards environmentally friendly agriculture. Despite the
natural phenomena playing hard against most crops, we need to gather all the
possible information on the plant–soil–water interactions in order to breed for this
century. Abiotic and biotic stresses will be targeted as most of the frontiers for
agriculture lie in nonoptimal areas, and genetic improvements through science will
play a major role in this conquer.
A. Costa de Oliveira (*)

Plant Genomics and Breeding Center, Eliseu Maciel School of Agronomy, Federal University of
Pelotas, Campus UFPel, Capão do Leão RS-96001-970, Brazil
e-mail: antonio.oliveira@pq.cnpq.br
R.K. Varshney
Centre of Excellence in Genomics (CEG), International Crops Research Institute for the Semi-
Arid Tropics (ICRISAT), Patancheru 502 324, A.P., India
Theme - Comparative and Applied Genomics, Generation Challenge Programme, c/o CIMMYT,
Int APDO, Postal 6-641, 06600 Mexico, DF, Mexico
School of Plant Biology (M084), The University of Western Australia, 35 Stirling Highway,
Crawley, WA 6009, Australia
e-mail: r.k.varshney@cgiar.org
A. Costa de Oliveira and R.K. Varshney (eds.), Root Genomics, 1

DOI 10.1007/978-3-540-85546-0_1, # Springer-Verlag Berlin Heidelberg 2011
2 A. Costa de Oliveira and R.K. Varshney
Root development, one of the major processes essential to the development of

flowering plants, remains poorly understood. Roots are a hidden part of plants for
many aspects and have not been the main subject of interest of researchers.
Nevertheless, roots play a major role in the plant–soil interactions, regarding
biological and physical aspects. The understanding of the physiological, molecular,
and developmental processes that roots undergo may represent a giant step on the
achievement of a more sustainable and energy-efficient agriculture. This book may
serve as a reference book in this context. Some concepts about root genomics
together with an overview on different chapters presented in this volume are
given in this article.
1.2 Root Genomics: An Overview
Root genomics research can be divided in the following four areas of research: (1) root
growth and development; (2) functional analyses of abiotic stress responses; (3)
functional analyses of biotic stress responses; and (4) quantitative trait loci (QTL)
analysis and molecular breeding. The understanding of basic mechanisms involving
root development and the interactions of roots and soils under various abiotic and
biotic stresses will pave the way for the next decades. Also, mutations obtained in
model species through the use of high throughput techniques such as TILLING
(targeted induced local lesions in genome) are turning root genomics an exciting
subject in plant molecular biology. An attempt has been made to cover all the above-
mentioned four areas of root genomics research.
1.2.1 Root Growth and Development
The breakthrough depiction of root development has started with Arabidopsis roots
(Dolan et al. 1993, 1994; Scheres et al. 1996). The events of division, enlargement,
and differentiation of cells in the roots are spatially separated. At the root tip, there
is a region of continuous cell division, the RAM (root apical meristem). The new
cells formed enlarge by a factor of 100-fold through a process of cell elongation.
After the cells reach a mature size, they differentiate into the various cell types of
the root. Root growth is accompanied by the formation of a series of lateral roots,
resulting in a branching pattern that covers higher volumes of soil space in every
step of branching. A range of root systems can be found in different plants including
from shallow patterns to very deep roots. Therefore, the identification of factors
affecting the patterns of root development is the major point in decoding the genetic
control of this organ.
In a paleontological context, the role of auxin in morphogenesis has allowed
the identification of vascular patterns preserved in fossils as records of auxin
gradients and growth dynamics (Boyce 2010). Roots evolved independently at least
1 Introduction to Root Genomics 3
in lycophytes and euphyllophytes (Gensel et al. 2001). Root traces have been found in
early Devonian soil horizons, contemporaneous with attached roots in lycophyte
related fossils. The presence of root hairs, root cap, and endogenous initiation shared
by roots has been proposed to have highly divergent origins (Boyce 2010). Shared
regulation by similar helix-loop-helix transcription factors (Menand et al. 2007)
suggests a homology between rhizoids and root hairs. The origin of root caps, on
the other hand, is suggested to be a response to the need of having a protective tissue to
the root apical meristem, a fast-growing region constantly in contact with a solid
surface, i.e., the soil. The appearance of adventitious roots may date the evolution of
endogenous initiation combined with reversed auxin transport, since the first appears
to have occurred repeatedly through times and is suggested to have been required for
the establishment of vascular continuity (Boyce 2005). Anatomical homogeneity/
heterogeneity is suggested as a reflection of stable/unstable environments faced by
land plants and epiphytes/swamp plants, respectively. Despite the environmental
differences, auxin transport mechanisms are thought to limit the anatomical variations
in roots (Boyce 2005; Raven and Edwards 2001).
Studying root development requires model species with simple root architecture.
Arabidopsis and rice are model species that have been fully sequenced and therefore
can provide good models for monocot and dicotyledoneous root development.
Arabidopsis root is composed of 15 distinct cell types arranged as concentric
cylinders around the radial axis (Iyer-Pascuzzi et al. 2009). MicroRNA-mediated
signaling has been reported to be involved in plant root development (Meng et al.
2010). Several of these miRNAs are interestingly shared by Arabidopsis and rice
despite their differences in root patterns and architecture. However, only a few genes
governing root development have been described in cereals, and differences between
monocots and dicots are quite remarkable when one regards at the root system.
Therefore, both models are necessary for the better understanding of the branching
patterns and functional specificities of roots. Two crown rootless mutants, crown-
rootless4 (crl4) and OsGnom1, affect the gene orthologous to GNOM1 in Arabidopsis
(Kitomi et al. 2008; Liu et al. 2009). GNOM1 is a membrane-associated guanine-
nucleotide exchange factor of the ADP-ribosylation factor G protein (ARF_GEF) that
regulates the traffic of PIN1 (PINFORMED 1) auxin efflux carrier proteins that
regulates auxin transport. GNOM1 is thought to be required for the formation of
the lateral primordium in Arabidopsis, by acting on the asymmetrical division of
pericycle cells (Coudert et al. 2010). Recently, a new notion on root system architecture
(RSA) has been described (Dorlodot et al. 2007). Root architecture importance for
plants lies in the fact that soil nutrients are not evenly distributed and the ability to
spatially deploy roots can constitute an advantage.
Developmental models could be an alternative to improve phenotyping in this
very plastic organ. Mapping the dynamics of roots per se or after inducing root
development under different stresses could bring better understanding and establish
genotype differences. Shoot-borne-root formation characterizes the difference
between cereals and the dicot model plant Arabidopsis. Several mutants that are
impaired in shoot-borne-root formation (4), lateral roots (4), primary root (6), and
root hairs (4) have been described in maize and rice (Hochholdinger et al. 2004).
Some of these genes controlling root development have been recently cloned and
will shed light on the influence of distinct root functions and architecture on grain
yield and performance in water-limited conditions (Hochholdinger and Tuberosa
2009). However, the overall trend is that single mutant standard analysis is shifting
to genome-wide approaches, leading to a speeding up of the process of generating
information. Proteomics- and metabolomics-generated datasets will need integration
with bioinformatics tools in order to translate the overwhelming amount of data into
biological meaningful phenomena.
1.2.2 Biotic Stress Tolerance
Biotic stress is caused by organism attacks to plants and can be caused by different
pathogens (virus, bacteria, or fungi) or pests (insects). Pathogen infections trigger
plant response mechanisms that are not restricted to the infection organ. The plant
senses the pest attack and responds with a range of different expressions of genes
regulating metabolites such as proteinase inhibitors, toxins, or volatiles that repel
pests or attract natural enemies. Herbivores or pathogens can elicit different types
of defense reaction. When vacuoles and trichomes are bursted as a consequence of a
chewing herbivore attack, compounds such as organic isothiocyanates can be
released (Bruce and Pickett 2007).
An interesting point of view is brought by on the cross-talk between shoot and
root (Van Dam et al. 2004; Bezemer and van Dam 2005). Induced responses are
complicated. The fact that hormone signaling pathways govern biotic and abiotic
stress responses is characterized by the fact that ABA is involved in many abiotic
responses and acts as a negative regulator of disease resistance (Fujita et al. 2006).
Other phytohormones, such as Salycilic acid (SA), Jasmonic Acid (JA), and
Ethylene (ET), play critical roles in biotic responses. Other responses are mediated
by MAP-kinase cascades, which control many biotic and abiotic responses. Other
evidence of this cross-talk is the presence of Reactive Oxygen Species (ROS) at
converging points between biotic and abiotic response pathways. The integration of
this network of responses is essential for the understanding of how roots participate
in this process and the intricate process of cross-signaling that this may need.
1.2.3 Abiotic Stress Tolerance
Roots are subjected to a wide range of stresses such as drought, flooding, salinity, as
well as nutrient starvation and metal toxicity such as Al, Cd, Fe, As, and Hg.
Cadmiun is a nonessential element for plants, its toxicity resulting in chlorosis and
stunting. Chlorosis seems to be an indirect effect on the uptake, transport, and use of
other elements such as Ca, Mg, Fe, Mn, Cu, Zn, P, and K. Cd also interferes with
hormones and disturbs plant water status, causing reduction of root hydraulic
conductivity, decrease of transpiration, and increase of stomatal resistance (Prasad

1995; Das et al. 1997; Aina et al. 2007). A proteomics approach revealed the
importance of two metabolic enzymes induced by 10 uM Cd that seems to play a
key role in the response to several abiotic stresses: alanine aminotransferase (ALT)
and Hexoquinase (HXK) suggest that these could be potential biomarkers for the
study of Cd toxicity (Aina et al. 2007). The accumulation of NaCl at root peripheral
regions limits growth by exerting osmotic and ionic stresses. Ionic stress is a
consequence of Naþ and Cl accumulation, disturbing the Kþ/Naþ ratio in the
plant cell (Hasegawa et al. 2000). Time-dependent effect of NaCl on the activities
of tonoplast proton pumps, showing distinct profiles for vacuolar proton transporting
ATPase and vacuolar proton transporting pyrophosphatase were reported. Activity
alterations were found to be due to posttranslational changes (Kabata and Ktobus
2008). The effects of salinity on Arabidopsis cells have been recently investigated
(Dinnenny et al. 2008). Transcriptional changes in response to salinity seem to be
highly constrained by developmental parameters. Iron deprivation and salt stress data
sets were compared. The largest set of coregulated genes displayed concerted down-
regulation in the epidermis and encoded genes important for protein biosynthesis.
Epidermis cells seem to present the least conserved patterns when different stresses
are applied (13–15%). A range of 244 genes are cell-type-specific and whose
expression pattern does not substantially change with stress. Chloroplast accumulation
was found to be a novel feature of the cortex in light-grown roots. Interestingly, rice
roots under excess iron stress seem to accumulate Rubisco peptides, as revealed by
proteomic studies (Costa de Oliveira, unpublished).
The responses of roots to abiotic stresses are though amenable to environmental
influences as well as cell-type. The high plasticity observed in the developmental
patterns plus the range of abiotic factors affecting root growth through the devel-
opment of plants picture a complex scenario composed of many players as well as
interactions among them.
1.2.4 QTL Analysis and Molecular Breeding
Root morphology is in most cases regulated by many genes with small effects and
highly influenced by the environment. Therefore, the study of root system related
genes will very often rely on QTLs analyses. A few examples on mapping and
identification of QTLs explaining the variation for root traits have become available
in some crop species (Price and Tomos 1997; Price et al. 2002; Giuliani et al. 2005).
Adventitious rooting has been considered to improve phosphorus uptake and deep
root growth to increase the ability to cope with drought (Ochoa et al. 2006; Macmillan
et al. 2006; Steele et al. 2006). In some cases, QTLs associated with root traits have
been cloned, e.g., root elongation in Arabidopsis (Sergeeva et al. 2006).
Although QTL analysis was developed to deal with environmental influence on
target characters, the high degree of plasticity presented by roots can mislead studies
and make it difficult to do a reliable phenotyping. However, at least in rice and
maize, QTL by environment interactions have been found to be weak, and marker-
assisted selection studies have been successful (Macmillan et al. 2006; Kamoshita
et al. 2002; Steele et al. 2006, 2007; Giuliani et al. 2005; Landi et al. 2005).
1.3 About the Book
This book covers all the four areas of research mentioned above. Some highlights of
the chapters included in this book are given below.
During the past decades, a considerable number of genes and gene networks have
been well described in the model species Arabidopsis thaliana. This knowledge can be
adapted for more complex plant systems as barley, rice, or maize. Despite their
agronomic importance, only a little is known about molecular basis of root formation
in crop species, and only few mutants together with corresponding genes have been
well characterized. In this context, Orman and colleagues from Silesian University,
Poland, have described the EST (expressed-sequence tag)-based approach, in Chap. 2,
to search for potential orthologous genes involved in root morphogenesis between
Arabidopsis, rice, and barley. The comprehensive gene list, developed by authors,
should provide strong platform for molecular studies and gene identification in barley
and related species.
Roots are exposed to a range of microbe, and there are several studies, as men-
tioned above, which deal with discussions on root–microbe interactions as well as
impact of biotic stresses on the root architecture. The Chap. 3, authored by Mathesius
and van Noorden from Australian National University, Australia, present the updates
on genomics of root–microbe interactions. Microbes influence roots by producing
signals, toxins, altering nutrient cycling, and by invading roots as endosymbionts or
endoparasites. Genomic tools have helped to elucidate the molecular changes induced
in roots by microbes. This chapter highlights some of the recent advances gained by
genomic and postgenomic studies to enhance knowledge in the area of root–microbe
interactions. Similarly, Deshpande and colleagues from Purdue University (USA),
University of Georgia (USA), Michigan Technological University (USA), and Instituto
Nacional de Tecnologı́a Agropecuaria (INTA, Argentina), in Chap. 4, discuss the
advances in the plant genetics for study of the roles of root exudates and microbes in
the soil. In order to dissect the relationships between soil microbes, plant exudates,
and plant function, authors planned to use host genetics to identify exudate::microbe
correlates that segregate with specific plant genes. Their studies indicated the great
potential for future investigations of the plant-determined chemical and organismal
diversity in the soil.
Abiotic stresses are the major stresses for limiting crop productivity in several crop
species, especially in developing countries. In majority of such cases, roots are the first
plant organs to be exposed as well as to respond. Some of these abiotic stresses in the
context of root genomics have been discussed in a few chapters. For instance, in
Chap. 5, Gruber and colleagues from Institut des Sciences du Végétal (ISV) and
Université Paris Diderot Paris 7 from France discuss the impact of abiotic stresses
such as drought and salt on the action and number of root meristems to determine root
architecture. In addition to Arabidopsis, authors have discussed recent results on
model legumes able to interact symbiotically with soil rhizobia to form new meris-
tems leading to the nitrogen-fixing nodule. Aluminum (Al) toxicity is another abiotic
stress that limits agricultural productivity over much of the world’s arable land by
inhibiting root growth and development. Affected plants have difficulty in acquiring
adequate water and nutrition from their soil environments and thus have stunted shoot
development and diminished yield. Hoekenga from US Department of Agriculture
(USDA) – Agricultural Research Station (ARS) (USA) and Magalhaes from
EMBRAPA Maize and Sorghum (Brazil) discuss in Chap. 6 the Al-tolerance
mechanisms. They propose and discuss the use of systems biology approaches to
study the mechanisms of Al tolerance and apply this knowledge to crop improvement
via marker-assisted breeding and translational genomics. Sousa and Costa de Oliveira
from Eliseu Maciel School of Agronomy, Campus UFPel (Brazil) discuss, in Chap. 7,
about root responses to other abiotic stresses such as soluble iron and short chain
organic acids in flooded soils, especially in the context of rice. Authors review the
progress on discovery of iron transporters as well as genetic variation present in rice
genotypes for flooding tolerance.
A number of studies have described QTLs that provide access to valuable
genetic diversity for the morphophysiological features that characterize root func-
tionality. Although a number of major QTLs have been identified as mentioned
above, none of these QTLs has been cloned so far in crop plants, mainly due to the
difficulty to accurately phenotype the target traits in a sufficiently large number of
plants. In this context, in Chap. 8, Tuberosa and colleagues present summary and
discuss the strategies for QTL cloning, especially in the context of maize. QTL
cloning should be facilitated by adoption of high-throughput phenomics platforms
as well as by information made available through genome and the profiling of the
transcriptome, proteome, and metabolome, all of which will contribute to the
identification of plausible candidate genes. Sheshashayee and colleagues from
University of Agricultural Sciences-Bangalore, India, in Chap. 9, have presented
phenotyping methodology for root traits and biotechnological approaches to
improve these roots traits with an objective of sustainable crop production. In
Chap. 10, Varshney and colleagues from ICRISAT, India, and Hokkaido University,
Japan, discuss the physiological and genomics approaches to dissect the root traits
at genetic and molecular level in context of devising the strategies for breeding for
root traits to enhance drought tolerance in chickpea. Authors have also discussed
the use of next generation sequencing technologies towards gene discovery and
marker development.
The last two chapters discuss the progress in the area of molecular breeding for root
traits for crop improvement. For instance, Raman from Wagga Wagga Agricultural
Institute, Australia, and Gustafson from University of Missouri, USA, in Chap. 11,
review the progress made on various aspects of molecular breeding for Al resistance
such as genetics, molecular mapping, comparative mapping, marker-assisted selec-
tion, candidate gene discovery and validation, and allele mining in key cereal crops
including wheat, barley, rice, maize, oats, sorghum, and rye. Similarly, Ismail and
Thomson from International Rice Research Institute, Philippines, in Chap. 12, have
summarized the progress made in unraveling molecular and physiological bases of
tolerance of various abiotic stresses encountered in rice problem soils including salt
stress and nutritional toxicities and deficiencies. Authors have also provided a brief
account of the progress towards developing and using marker-assisted back crossing
(MABC) for cultivar improvement in rice.
1.4 Concluding Remarks
The field of root genomics is an exciting and promising field of research. Some of
these areas of research have been detailed in some chapters of the book. The
technical advances in plant-omics are prone to generate enough data to push
forward the science of root genomics. Candidate gene identification is a strategy
that is getting stronger every year. The production of genomic sequences from
many sequencing projects is making the availability of specific genes more
frequent. Bioinformatic tools and reverse genetic approaches such as TILLING,
gene knockout mutants, or RNAi are prone to increase the success in this strategy
(Dorlodot et al. 2007). An ever neglected part of the plant, roots seem to hold the
key for the next plant breeding revolution, leading to improved crop productivity
in environmentally challenged situations.
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Chapter 2
EST-Based Approach for Dissecting Root
Architecture in Barley Using Mutant Traits
of Other Species
Beata Orman, Aleksander Ligeza, Iwona Szarejko, and Miroslaw Maluszynski
Contents
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2 Root Mutants of Arabidopsis Published in Pubmed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.3 Root Mutants in Monocotyledonous Species Published in Pubmed . . . . . . . . . . . . . . . . . . . . . 42
2.4 Strategy for EST Data-Mining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.4.1 Searching for Potential Orthologs Between Arabidopsis and Barley . . . . . . . . . . . . 46
2.4.2 Arabidopsis and Rice Genes Comparisons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
2.4.3 Searching for Potential Orthologs Between Other
Monocotyledons and Barley . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
2.4.4 Phylogenetic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
2.4.5 Synteny Detection in Arabidopsis and Rice Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2.5 In Silico vs. Laboratory Approach to Gene Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.6 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
2.6.1 Rice and Arabidopsis Searches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
2.6.2 Sequence Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
2.6.3 ESTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
2.1 Introduction
There are increasing evidences that root architecture is a fundamental aspect of

plant growth. The role of root system includes acquisition of water and nutrients,
anchorage of the plant in the soil, synthesis of hormones, and also storage functions.
It was generally considered that root characteristics could be important for breed-
ing, to obtain genotypes of a higher adaptability to unstable soil and climatic
conditions (Gorny 1992; De Dorlodot et al. 2007) and higher productivity (Lynch
1995). Despite their importance, little is known about genetic basis of root system
formation and architecture in major crop species. A great progress in understanding
the molecular processes underlying root development has been achieved only in
Arabidopsis thaliana (Scheres et al. 2002; Casimiro et al. 2003; Casson and
B. Orman, A. Ligeza, I. Szarejko (*), and M. Maluszynski

Department of Genetics, Silesian University, Katowice, Poland
e-mail: iwona.szarejko@us.edu.pl

12 B. Orman et al.
Lindsey 2003; Ueda et al. 2005; Zhang et al. 2007; Busov et al. 2008). This progress
was accomplished through detailed analysis of root mutants with the use of
advanced molecular, genomic, and bioinformatic tools available for Arabidopsis.
Recently, several root mutants have been reported in three cereal species, rice (Ma
et al. 2001; Zimmer et al. 2003; Liu et al. 2005; Inukai et al. 2005; Jiang et al. 2005;
Li et al. 2006a; Kim et al. 2007), maize (Lim et al. 2005; Woll et al. 2005; Wen et al.
2005; Hochholdinger et al. 2008), and wheat (Wang et al. 2006). Some of them
have become the subject of studies similar to Arabidopsis that have led to the
identification of homologous and novel genes controlling root system formation in
monocotyledons (Morita and Kyozuka 2007). There is, however, a lack of similar
knowledge in barley. These differences in progress of knowledge between mono-
cotyledonous and dicotyledonous species could be considered as a result of the
more extensive size of adult cereal root systems and lack of such efficient screening
strategies like those developed for Arabidopsis. Based on this, we will focus on root
development in monocotyledons, especially in barley, which is the fourth most
important crop in the world after maize, wheat, and rice. Recently, it is becoming a
novel cereal model plant because of its true diploidy (Sreenivasulu et al. 2008).
Root system of monocotyledonous plants is generally composed of two funda-
mental parts: seminal root system, which develops from initials present in embryo,
and nodal (often called adventitious or shoot-borne) root system, which originates
from shoot (Hackett 1968). The dicotyledonous species develop a taproot system
with one primary root and lateral branches, which remain active during the whole life
cycle. However, dicotyledonous plants can also form roots called “adventitious”
under unusual circumstances such as wounding or hormone application, etc., at
uncharacteristic sites on a plant. Following Hochholdinger and coworkers (2004),
we also suggest not calling monocotyledonous stem-derived crown and brace roots
“adventitious” because they belong to the normal developmental program of cereals.
Despite having to fulfill the same fundamental functions, the root systems of mono-
cotyledons and dicotyledons differ both in morphology and anatomy. In monocoty-
ledons, the secondary root growth do not occur, and root vessels are relatively
uniform cylinders (in the absence of environmental stimuli) (Gorny 1992). The
adult crop plant exhibits an extensive shoot-born root system, which plays a major
role in the postembryonic root architecture (Hochholdinger et al. 2004; Hochholdin-
ger and Zimmermann 2008). Nevertheless, it has been reported that maize seminal
roots have relatively high water uptake capacity compared to other root types, which
makes them important throughout whole plant life (Osmont et al. 2007).
2.2 Root Mutants of Arabidopsis Published in Pubmed
Both forward and reverse genetic approaches have been used to increase knowledge
about root architecture. As there are many mutagenesis methods, the use of
chemical mutagenesis mostly by EMS and insertional mutagenesis using T-DNA
insertion, followed by mutant screening, apparently dominates. Using EMS, 147
gene alleles were obtained, 140 alleles by insertional mutagenesis (e.g., 19 by
2 EST-Based Approach for Dissecting Root Architecture in Barley Using Mutant Traits 13
transposable elements, 118 by T-DNA, 2 by promoter trap and 1 by activation

tagging), whereas 22 alleles were obtained by physical approach (nine by fast
neutrons, six by X-ray, seven by gamma rays). Reverse approach (e.g., RNAi,
overexpression) were also commonly used to study influence of a gene of interest
on root traits.
Using these strategies, it was possible to build the model pattern of root devel-
opment in dicotyledons, based on data from reference Arabidopsis. Up to now,
many genes have been shown to be involved in various aspects of Arabidopsis root
development (Tables 2.1 and 2.2). Many of them have a pleiotropic effect not only
on various stages of root development but also on whole plant per se. Nevertheless,
we divided Arabidopisis genes controlling root system into formation of radial and
longitudinal pattern, keeping in mind that assigning genes to only one chosen
category could be misleading. The Arabidopsis radial pattern consists of a number
of defined cell types organized in concentric layers, with the epidermis, ground
tissue composed of cortex and endodermis, and the last main part called stele, which
includes pericycle surrounding the central vascular cylinder (Scheres et al. 2002;
Casson and Lindsey 2003). Based on this, we secondly divided genes responsible
for root radial pattern into three groups, which assemble genes involved in epidermis,
ground tissue, and stele development.
The first one (Table 2.1) includes genes involved in root hair development as a
specific product of root epidermis. Both monocotyledonous and dicotyledonous
root systems increase absorptive surface through the formation of root hairs. In
Arabidopsis, root hairs always form on epidermal cells positioned over the radial
cell wall between cortical cells (Dolan and Costa 2001). However, it is difficult to
predict root hair-forming epidermal cells in cereals (Hochholdinger et al. 2004). In
Arabidopsis, epidermis is composed of trichoblasts, which develop into root hair
cells, and atrichoblasts, which remain hairless. The identity of these cells is
regulated by positional information – hair-forming cells are located above two
underlying cortical cells. The genetic analysis of root hair development has identi-
fied at least 39 genes that are required for the initiation and growth of the root hair.
Some of them, such as TRANSPARENT TESTA GLABRA1 (TTG1), GLABRA3
(GL3), ENHANCER OF GLABRA3 (EGL3), and GLABRA2 (GL2), have been
well described (Galway et al. 1994; Walker et al. 1999; Bernhardt et al. 2003).
Both TTG1 and GL2 mutants have root hairs at nearly all root epidermal cells
(Walker et al. 1999; Ohashi et al. 2003), whereas GL3 and EGL3 mutants have
reduced numbers of atrichoblasts (Bernhardt et al. 2003). TTG1 encodes a protein
with WD40 repeats (Mendoza and Alvarez-Buylla 2000), which is localized in the
nuclei of trichomes at all developmental stages (Zhao et al. 2008). It seems that GL2
is a direct target of GL3 and EGL3, whereas TTG1 is directly regulated by GL1
(Zhao et al. 2008).
The second group includes genes responsible for ground tissue pattering, com-
posed of one cortex and one endodermis layer (Table 2.1), which originate from the
common initial cell adjacent to the quiescent center (QC) (Scheres et al. 2002).
Outside the endodermis, there are 4–6 layers in barley (Jackson 1922) and 8–15 in
rice and corn (Hochholdinger et al. 2004) of bigger and thin-walled loosely packed
Table 2.1 Mutated genes responsible for Arabidopsis root radial pattern
14
Gene name (alias) Accession Allele/mutation strategy/ Mutant phenotype References

number reverse approach
Root hairs
TRANSPARENT TESTA AT5G24520 ttg-1/EMS All cells with root hairs Galway et al. (1994), Walker et al.
GLABRA1 (TTG1) (1999)
GLABRA2 (GL2) AT1G79840 p777/T-DNA insertion All cells with root hairs Ohashi et al. (2003)
WEREWOLF (WER) AT5G14750 wer-1/EMS All cells with root hairs Lee and Schiefelbein (1999)
CAPRICE (CPC) AT2G46410 cpc-1/T-DNA insertion All cells without root hairs Wada et al. (1997)
GLABRA3 (GL3) AT5G41315 gl3-1/EMS Reduced number of atrichoblasts Bernhardt et al. (2003)
(much more root hairs)
ENHANCER OF AT1G63650 egl3-77439/T-DNA Reduced number of atrichoblasts Bernhardt et al. (2003)
GLABRA3 (EGL3) insertion (much more root hairs)
ENHANCER OF AT1G01380 EMS All cells without root hairs or root hairs are Kirk et al. (2004)
TRIPTICHON AND very sporadic
CAPRICE1 (ETC1)
ETROPIC ROOT HAIR 1 ? erh1/fast neutrons Reduced number of atrichoblasts (much Hauser et al. (1995), Schneider
(ERH1) more root hairs) et al. (1997)
ETROPIC ROOT HAIR3 AT1G80350 Gamma rays Reduced number of atrichoblasts (much more Hauser et al. (1995), Schneider
(ERH3) root hairs) et al. (1997)
TORNADO1 (TRN1) AT5G55540 trn1-1/T-DNA insertion Radial pattern is unsettled, pattern of root Dolan (2000)
hairs is twisted like DNA helix
TORNADO2 (TRN2) AT5G46700 trn2-2/EMS Radial pattern is unsettled, pattern of root Dolan (2000)
hairs is twisted like DNA helix
ROOT HAIRLESS 1 AT1G48380 rhl1-1/T-DNA insertion Root hairs very sporadic, pattern of Schneider et al. (1997, 1998)
(RHL1) rhl1-2/unknown trichoblasts and atrichoblasts unsettled
ROOT HAIRLESS AT5G02820 T-DNA insertion Root hairs very sporadic, pattern of Schneider et al. (1997)
2 (RHL2) trichoblasts and atrichoblasts unsettled
ROOT HAIRLESS 3 AT3G20780 rhl3-1/EMS Root hairs very sporadic, pattern of Schneider et al. (1998)
(RHL3) trichoblasts and atrichoblasts unsettled
CONSTITUTIVE TRIPLE AT1G01380 ctrl-6/T-DNA insertion Root hairs are formed on other place than Kieber et al. (1993), Dolan et al.
RESPONSE (CTR1) usually (1994)
B. Orman et al.
AT1G66340 etr1-1/EMS Masucci and Schiefelbein (1996)

ETHYLENA RECEPTOR Root hairs are formed near to the basal part of
1 (ETR1) cell
ETHYLENE AT3G51770 eto1-1/EMS Root hairs are formed near to the apical part Masucci and Schiefelbein (1996),
OVERPRODUCER 1 of cell Yoshida et al. (2006)
(ETO1)
ROOT HAIR ? EMS Root hairs are very sporadic and formed near Masucci and Schiefelbein (1994),
DEFECTIVE 6 to the basal part of cell, more than one Dolan (2001)
(RHD6) root hair on one cell
SALT OVERLY AT5G37850 sos4-1/EMS Root hairs are very, very sporadic Shi and Zhu (2002)
SENSITIVE 4 (SOS4)
ROOT HAIR AT1G64440 rhd1-2/EMS Primordium is very big, root hairs with Schiefelbein and Somerville (1990)
DEFECTIVE 1( rhd1-1/EMS normal length
RHD1)
TIP GROWTH AT5G20350 tip1-1/EMS Primordium is bigger, root hairs are shorter Ryan et al. (1998)
DEFECTIVE 1 (TIP1) and often branched, sometimes there are
2–4 root hairs on one cell
SUPERCENTIPEDE 1 ? EMS 1–5 primordiums on one cell Grierson et al. (2001)
(SCN1)
TINY ROOT HAIR 1 AT4G23640 trh1/EMS Root hair growth stopped at primordium Rigas et al. (2001), Vicente-Agullo
(TRH1) stage et al. (2004)
HAIR DEFECTIVE AT5G51060 rhd2-1/EMS Root hair growth stopped at primordium Schiefelbein and Somerville (1990)
2 (RHD2) stage
SHAVEN1,2,3 (SHV1,2,3) ? EMS Root hairs are shorter Parker et al. (2000)
KOJAK (KJK) AT3G03050 csld3-1/T-DNA insertion Root hairs rupture at their tip soon after Favery et al. (2001)
initiation
MRH2 AT3G54870 mrh2-1/T-DNA insertion Mutant exhibits wavy and branching root hair Yang et al. (2007)
phenotype
LRR/EXTENSIN 1 (LRX1) AT1G12040 lrx1/En-1 transposition Root hairs are shorter, often branched Baumberger et al. (2001)
DEFORMED ROOT ? EMS Root hairs are shorter, primordium is bigger, Ringli et al. (2002)
HAIRS 1 (DER1) and sometimes there are 2 root hairs on
2 EST-Based Approach for Dissecting Root Architecture in Barley Using Mutant Traits
one cell
AT2G35630 EMS Root hairs are wavy and branched Whittington et al. (2001)
15
(continued)
Table 2.1 (continued)
16

MICROTUBULE
ORGANIZATION 1
(MOR1)
INCOMPLETE ROOT AT5G62310 T-DNA insertion Root hairs are shorter Oyama et al. (2002)
HAIR ELONGATION
(IRE)
ROOT HAIR AT3G13870 rhd3-1/EMS Root hairs are shorter and wavy Schiefelbein and Somerville
DEFECTIVE 3 (1990), Galway et al. (1997),
(RHD3) Zheng et al. (2004)
ROOT HAIR ? EMS Root hairs are shorter and wavy Schiefelbein and Somerville (1990)
DEFECTIVE 4
(RHD4)
CAN OF WORMS 1 AT4G34580 T-DNA insertion Root hairs are shorter, wavy, and 1–3 on one Grierson et al. (1997), B€ohme et al.
(COW1) cell (2004)
BRISTLED1 (BST1) AT5G65090 Fast neutrons Root hairs are shorter, wavy and branched Parker et al. (2000)
CENTIPEDE 1,2,3 ? EMS Root hairs are shorter, wavy and branched Parker et al. (2000)
(CEN1,2,3)
ACTIN 2 (ACT2) AT3G18780 act2-3/T-DNA insertion Root hairs are shorter and branched Ketlaar et al. (2003)
SUPRESOR OF AUXIN AT2G33120 EMS Root hairs are longer and on almost all cells Cernac et al. (1997)
RESISTANCEL
(SAR1)
ROOT AND POLLEN AT2G35210 T-DNA insertion Aberrant root hair phenotype, including Song et al. (2006)
ARFGAP (RPA) bulged, branched and shorter root hairs
Ground tissue pattern (cortex þ endodermis)
POM-POM1 (POM1) AT1G05850 pom 1-1/T-DNA insertion Shorter root and significantly greater cell Hauser et al. (1995), Scheres et al.
volume (2002)
POM-POM2 (POM2) ? pom2-1, 2-2/fast neutron Shorter root and significantly greater cell Hauser et al. (1995), Scheres et al.
volume (2002)
? qui-1/X-ray Shorter root and significantly greater cell Hauser et al. (1995), Scheres et al.
B. Orman et al.
qui-2/T-DNA insertion volume, decreased cell elongation, (2002)

PROCUSTE1/QUILL/ qui-3/EMS specifically in roots and dark-grown
ATCESA6 (PRC1/ hypocotyls
QUI)
COBRA (COB) AT5G60920 cob1-4/EMS Abnormal root cell expansion, greatest in Benfey et al. (1993, Scheres et al.
cob-2/X-ray the epidermal cells (2002), Roudiera et al. (2005)
cob-2/T-DNA insertion
SHORT ROOT (SHR) AT4G37650 shr-1/T-DNA insertion Determinate root growth, very short root Benfey et al. (1993), Scheres et al.
missing an internal cell layer, mutant (2002), Franco-Zorrilla et al.
layer has attributes of cortex only (2005)
SCARECROW (SCR) AT3G54220 scr-4, scr-1/T-DNA Defects in the division and/or specification Benfey et al. (1993), Scheres et al.
insertion of endodermis and cortex (2002)
KORRIGAN (KOR) AT5G49720 kor1-1/EMS Radially expanded hypocotyl cells, impaired Zuo et al. (2000), Scheres et al.
kor1-2/Agrobacterium root expansive growth, formation of (2002)
transformation aberrant cell plates, incomplete cell walls,
and multinucleated cells, leading to
severely abnormal root morphology (cells
divided randomly and often contained
incomplete cell walls
LION’S TAIL ? T-DNA insertion Abnormal root cell expansion, greatest in Benfey et al. (1993), Hauser et al.
the stele cells (1995), Scheres et al. (2002)
SABRE (SAB) AT1G58250 EMS Abnormal root-cell expansion, primarily in Benfey et al. (1993)
radial orientation. Expansion greatest in
cortex cells
FACKEL (FK) AT3G52940 T-DNA insertion Short roots and hypocotyl, defective cell Souter et al. (2002)
shape, supernumerary cell layers and
aberrant vascular patterning
HYDRA 1 (HYD1) AT1G20050 hyd1-2/ T-DNA insertion Short roots and hypocotyls, defective cell Souter et al. (2002)
shape, supernumerary cell layers and
aberrant vascular patterning. Root may
cease cell division within 2 weeks after
germination, or may continue to grow

very slowly until the seedling dies
17
(continued)
18

CUDGEL-1 ? cud-1/X-ray Shorter root and significantly greater cell Hauser et al. (1995), Scheres et al.
volume (2002)
SCHIZORIZA (SCZ) ? Ac/Ds Subepidermal layer (ground tissue) develops Mylona et al. (2002)
root hairs – supernumerary layers in the
ground tissue
KNOPF (KNF) AT1G67490 knf-14/EMS Radially swollen root phenotype due to Gillmor et al. (2002)
cellulose deficiency and isotropic embryo
growth
RADIAL SWELLING 1 AT4G32410 rsw1-1, 1-2/EMS Radially swollen root phenotype due to Gillmor et al. (2002)
(RSW1) cellulose deficiency and isotropic embryo
growth
RADIAL SWELLING 3 AT5G63840 rsw3-1/EMS Temperature-sensitive, cellulose-deficient Burn et al. (2002)
(RSW3) mutant with radially swollen roots
RADIAL SWELLING 4 ? EMS Radially swolling roots and temperature Wiedemeier et al. (2002)
(RSW4) sensitive phenotype. Cortical
microtubules and cellulose microfibrils
are neither depleted nor disoriented
RADIAL SWELLING 7 ? EMS Radially swollen roots and temperature Wiedemeier et al. (2002)
(RSW7) sensitive phenotype. Cortical
microtubules and cellulose microfibrils
are neither depleted nor disoriented
PLEYADE (PLE) AT5G51600 ple-1, -2/EMS, ple-3/T- Shorter roots exhibit a wavy growth pattern Műller et al. (2002)
DNA and develop more lateral roots. Irregular
cell expansion, multinucleated cells, cell
wall stubs, epidermis, cortex and
endodermis are radially enlarged;
symmetry of the vascular tissues is
disrupted and synchronized cell divisions
in incompletely separated cells that are all
characteristics of defective cytokinesis
B. Orman et al.
HYADE1 (HYA1) ? hya-1, -2, -3/EMS Shorter roots exhibit a wavy growth pattern Műller et al. (2002)
and develop more lateral roots. Irregular
cell expansion, multinucleated cells, cell
wall stubs, epidermis, cortex and
endodermis are radially enlarged;
symmetry of the vascular tissues is
disrupted and synchronized cell divisions
in incompletely separated cells that are all
characteristics of defective cytokinesis
BOTERO1 (BOT) ? bot1-1, 1-3, 1-4, 1-5/EMS Shorter and thicker root and hypocotyl. Bichet et al. (2001)
bot1-2/retrotransposon Tnt1 Affected in anisotropic growth, loosely
bot1-7, 1-8/T-DNA organized microtubules
insertion
CLUB ? EMS Lack of primary roo. Cell wall stubs, gapped S€ ollner et al. (2002)
walls and multinucleate cells. Incapable
of growing long root hairs, likely to
represent a tip growth defect.
BUBLINA ? EMS Lack of primary root. Cell wall stubs, gapped S€ ollner et al. (2002)
walls and multinucleate cells. Long root
hairs.
BIMS ? EMS Lack of primary root. Cell wall stubs, gapped S€ ollner et al. (2002)
walls and multinucleate cells. Long root
hairs.
MASSUE ? EMS Stunted root, cell wall stubs, gapped walls S€
ollner et al. (2002)
and multinucleate cells
BLOATED ? EMS Stunted root, cell wall stubs, gapped walls S€
and multinucleate cells. Long root hairs
ROD ? EMS Stunted root, cell wall stubs, gapped walls S€
KEULE AT1G12360 EMS Lack of primary root. Cell wall stubs, gapped S€ ollner et al. (2002)
walls and multinucleate cells. Incapable

(continued)
19
20

of growing long root hairs, likely to
represent a tip growth defect.
KNOLLE AT1G08560 X-ray Lack of primary root. Cell wall stubs, gapped S€ollner et al. (2002)
walls and multinucleate cells. Cytokinesis
defects are more severe than in keule
mutants: severely perturbed epidermis
and long root hairs
HINKEL (HIK) AT1G18370 EMS Long root hairs, cell wall stubs, gapped walls S€
SHORT BLUE ROOT ? EMS Deformation of epidermal cells, larger Subramanian et al. (2002)
(SBR) meristematic cells, disorganized root
vascular tissue. Reduced lateral root
initiation, adventitious roots often form
on hypocotyl
Stele pattern (pericycle þ vasculature)
ALTERED PHLOEM AT1G79430 En-1 transposition Defects in vascular tissue Bonke et al. (2003)
DEVELOPMENT
(APL)
LONESOME HIGHWAY ? EMS Lack of root bilateral symmetry: reduced the Ohashi-Ito and Bergmann (2007)
(LHW) number of cells in the center of the root,
single xylem and phloem poles
WOODEN-LEG (WOL) AT2G01830 ahk4-1/ T-DNA insertion Protoxylem is the only tissue in the vascular Scheres et al. (2002), Sieberer et al.
wol-1/ EMS cylinder (2003), Franco-Zorrilla et al.
(2005)
KOBITO 1 (KOB1) AT3G08550 eld1-1/gamma rays Cellulose-deficient dwarf mutant. Pagant et al. (2002)
Randomized microfibrils occluded by a
layer of pectic material
B. Orman et al.
IRREGULAR XYLEM 1 AT4G18780 irx1-1/ EMS Severe deficiency in the deposition of Taylor et al. (2000)
(IRX1) cellulose in secondary cell walls, which
results in collapsed xylem cells
irx1-5/ T-DNA insertion
IRREGULAR XYLEM 3 AT5G17420 irx3-1/ EMS Severe deficiency in the deposition of Taylor et al. (2000)
(IRX3) cellulose in secondary cell walls, which
results in collapsed xylem cells
ECTOPIC ? EMS Mutant exhibits altered patterns of Caño-Delgado et al. (2000)
LIGNIFICATION 1 lignification (ectopic lignification),
(ELI-1) stunted phenotype and disorganized
xylem tissue
21
22 B. Orman et al.
cortical cells (Briggs 1978), whereas in Arabidopsis, root comprises only one
endodermis and one cortical layer (Scheres et al. 2002). The one layer of endoder-
mis is exceptionally thick-walled, just like that reported earlier in rice, maize, and
onion (Jackson 1922) with a “Caspian strip” in the walls (Karas and McCully
1973). Many mutations that disrupt patterning of the ground tissue have been
identified. For example, both the SCARECROW (SCR) and SHORT ROOT (SHR)
mutants have a single layer instead of cortex and endodermis. These genes encode
putative transcription factors of the GRAS family responsible for specifying QC
and for controlling the periclinal cell division of the daughter cell of their common
initial cell, which leads to two adjacent layers (Ueda et al. 2005). However, SCR
mutant layer has differentiated attributes of both cortex and endodermis, whereas
SHR layer attribute only to cortex (Scheres et al. 2002). SCR was previously shown
to act downstream of SHR (Ueda et al. 2005), whereas Levesque and coworkers
(2006) suggested that SHR not only directly regulates the transcription of SCR
through binding to the chromatin upstream of the gene but also functions in
development of the vascular tissue.
In the middle of the young barley root is a duct bordered by thin-walled cells,
which becomes thickened during aging. The continuity of one layer of pericycle
cells is broken by the xylem groups, which contain large vessels. The number of
xylem groups in barley root is from 6 to 8 alternating with groups of phloem
(Jackson 1922). Protoxylem elements abut directly to the single layer of endoder-
mis, the walls of which thicken with age (Briggs 1978). Fully developed monocot-
yledonous root consists of much more thickened cell walls in stele, and
sclerenchyma develops in the outer cortex (Briggs 1978). In contrast to monocoty-
ledonous root radial pattern, the primary vascular pattern in Arabidopsis roots
involves a xylem axis and two phloem poles, surrounded by one pericycle layer
(Scheres et al. 2002). Only few Arabidopsis genes, which are responsible for stele
pattern, have been described (Table 2.1). In the WOODEN-LEG (WOL) mutant,
protoxylem is the only tissue in the vascular cylinder (Sieberer et al. 2003). It has
been shown that this gene encodes a cytokinin receptor (Franco-Zorrilla et al.
2005), which is required for asymmetric cell divisions of phloem and procambium
initial cells (Scheres et al. 2002). Defects in vascular tissue could be also observed
in ALTERED PHLOEM DEVELOPMENT (APL) mutant. This gene, which encodes
a MYB transcription factor, has a dual role both in promoting phloem differentia-
tion and in repressing xylem differentiation during vascular development (Bonke
et al. 2003).
Root meristem tissues are organized in longitudinal cell files. From the root tip to
the plant base, three main regions could be distinguished: the division, elongation,
and the differentiation zone (Table 2.2). During both monocotyledons and dicoty-
ledons embryogenesis, first the primary or embryonic radicle and few seminal roots
are formed, respectively, whereas lateral roots (LRs) originate from existing roots
postembryonically. LRs originate from the group of pericycle cells in Arabidopsis
(Malamy and Benfey 1997; Scheres et al. 2002), whereas in monocotyledons,
endodermis is also involved (Hochholdinger et al. 2004; Karas and McCully
1975). In Arabidopsis, lateral roots emerge from the pericycle cells adjacent to
Table 2.2 Mutated genes responsible for Arabidopsis root longitudinal pattern
Gene name (alias) Accession Allele/mutation Mutant phenotype References
number strategy/reverse
approach
Meristematic zone
ROOT PRIMORDIUM DEFECTIVE 1 AT4G33495 rpd1-1/EMS Temperature-sensitive mutant with defects at the Konishi and Sugiyama
(RPD1) initial stage of root primordium development. (2006)
Embryogenesis arrested at the globular stage
HALTED ROOT (HLR) AT4G29040 hlr-1, hlr-2/T-DNA In postembryonic meristems the cellular Ueda et al. (2004)
insertion organization is disrupted, the activity of
proteasomes is reduced
RUB1 CONJUGATING ENZYME 1 AT4G36800 T-DNA insertion Dwarf phenotype. Reduced response to the change in Dharmasiri et al. (2003)
(RCE1) the gravity vector deficient in auxin and
jasmonate response, fewer lateral roots in
response to auxin
PLETHORA 1 (PLT1) AT3G20840 plt1-1, 1-2, 1-3, 1-4, Mutant shows an abnormal cellular organization of Aida et al. (2004)
1-5 /T-DNA the hypophyseal derivatives
insertion
PLETHORA 2 (PLT2) AT1G51190 plt2-2/ T-DNA Mutant shows an abnormal cellular organization of Aida et al. (2004)
insertion the hypophyseal derivatives
GNOM/EMB30 (GN) AT1G13980 emb30-1, emb30-2, Failure in maintenance of primary root meristem Shevell et al. (2000),
gn/EMS activity; reduced LR number Geldner et al. (2003)
STEROL METHYLTRANSFERASE 1 AT5G13710 EMS Mutants displays several conspicuous cell polarity Willemsen et al. (2003)
(SMT1) defects, primary and lateral roots are shorter
FASS (FS) AT5G18580 EMS Drastically changed the shape of the seedling without Torres-Ruiz and J€urgens
altering body pattern and affected cell elongation (1994)
and orientation of cell walls
HOBBIT (HBT) AT2G20000 EMS Postembryonic meristem activity is absent and the Blilou et al. (2002),
distal cell types (QC, columella- and lateral root Scheres et al. (2002)
cap) do not differentiate. The earliest defect
found in mutants is disturbance of cell division

planes in the hypophysis, the progenitor cell for
23
the QC and columella

(continued)
24

approach
BODENLOS (BDL) AT1G04550 EMS Mutant failure to establish the hypophysis which Hamann et al. (1999,
caused severe primary root defects 2002), Scheres et al.
(2002)
MONOPTEROS (MP) AT1G19850 EMS Mutant failure to establish the hypophysis which Hamann et al. (2002)
caused severe primary root defects (loss-of-
function)
ACID-INDUCED PROTEIN 13 AT2G33310 EMS Lack of root caused by failure in the specification Weijers et al. (2005)
(IAA13) of the hypophysis and subsequent abnormal cell
division patterns
ROOT MERISTEMLESS 1 (RML1) AT4G23100 EMS Extremely short mature root (1–2 mm) composed of Vernoux et al. (2000)
the same number of cells and cell files as the
embryonic root, unable to establish and maintain
an active, undifferentiated meristematic zone
(mutation does not affect axial and radial patterns
of root cell organization). Mutant produces lateral
roots readily
ROOT MERISTEMLESS 2 (RML2) ? EMS Extremely short mature root (1–2 mm). Mutant Cheng et al. (1995)
produces nodule-like structures but not lateral
roots. Limited number of cell divisions
HISTIDINOL-PHOSPHATE AT1G71920 emb-2196/ T-DNA Very short root system, unable to sustain primary Mo et al. (2006)
AMINOTRANSFERASE (HPA) insertion hpa1/ root growth 2 days after germination
EMS
INCURVATA 4 (ICU4) AT1G52150 icu4-1, icu4-2/En- Longer root hairs, higher number of secondary roots, Ochando et al. (2006)
2 transposition reduced root length and an aberrant cell pattern in
icu4-3, icu4-4/ the root apical meristem
T-DNA insertion
TEBICHI (TEB) AB192295 teb-1/T-DNA Short root, split root tip and an aberrant pattern of Inagaki et al. (2006)
insertion cell division in postembryonic development
TONSOKU (TSK) AT3G18730 T-DNA insertion Short roots and altered responses to DNA damage Inagaki et al. (2006)
B. Orman et al.
RETINOBLASTOMA-RELATED AT3G12280 rbr1-3/ T-DNA Supernumerary stem cells Wildwater et al. (2005)
(RBR) insertion
AUXIN INFLUX 1 (AUX1) AT2G38120 aux1110, aux12, 50% reduction in number of LR primordia, reduced Casimiro et al. (2003)
aux1106, aux17, auxin-sensitive root elongation, perturbed in
aux122/EMS, gravitropism
aux121/X-ray
PIN-FORMED 1 (PIN1) AT1G73590 En-1 transposition Defects in auxin transport. Reduction of root length, Friml et al. (2003)
meristem size and root elongation zone size
PIN-FORMED 2 (PIN2) AT5G57090 En-1 transposition Defects in auxin transport. Reduction of root length, Muller et al. (1998)
meristem size and root elongation zone size
PIN-FORMED 3 (PIN3) AT1G70940 pin3-5/ T-DNA Defects in auxin transport. Reduction of root length, Friml et al. (2003)
insertion meristem size and root elongation zone size.
Subtle cell division defects in the QC and
columella root cap
PIN-FORMED 4 (PIN4) AT2G01420 pin4-3/transposon Defects in auxin transport. Reduction of root length, Friml et al. (2003)
insertion meristem size and root elongation zone size.
Subtle cell division defects in the QC and
columella root cap
PIN-FORMED 7 (PIN7) AT1G23080 pin7-1, pin7-3/ Defects in auxin transport. Reduction of root length, Friml et al. (2003)
transposon meristem size and root elongation zone size.
insertion Subtle cell division defects in the QC and
pin7-2/T-DNA columella root cap
insertion
PINOID (PID) AT2G34650 pid-1, pid-2/EMS Mutants do not display a root phenotype. Shishkova et al. (2008)
Constitutive overexpression results in a
consumption of the primary root meristem within
a few days after germination: all cells at the root
tip become elongated and root hairs cover the
primary root tip
ISOPENTENYLTRANSFERASE 3 AT3G63110 atipt 3/ T-DNA Triple cytokinin biosynthetic mutant with severely Dello-Ioio et al. (2007)
(IPT3) insertion reduced cytokinin level. Enlarged RAM shows an

increased number of meristematic cells
25
(continued)
26

approach
ISOPENTENYLTRANSFERASE AT5G19040 atipt 5/T-DNA Triple cytokinin biosynthetic mutant with severely Dello-Ioio et al. (2007)
ISOPENTENYLTRANSFERASE AT3G23630 atipt 7/T-DNA Triple cytokinin biosynthetic mutant with severely Dello-Ioio et al. (2007)
ENDO-BETA-1,4-GLUCANASE ? T-DNA insertion Mutant forms the root cap and sheds root cap cells Campillo et al. (2004)
(CEL5) but sloughing is less efficient compared to wild
type
NO HYDROTROPIC RESPONSE ? EMS No positive hydrotropic response. Abnormal root cap Eapen et al. (2003)
(NHR) morphogenesis and reduced root growth
sensitivity to abscisic acid (ABA) and the polar
auxin transport inhibitor N-(1naphtyl) phtalamic
acid (NPA). Homozygous condition results in a
lethal phenotype
MIZU-KUSSEI 1 (MIZ1) ? EMS Mutant impaired in hydrotropism but shows normal Kobayashi et al. (2007)
gravitropism and elongation growth
SKU 5 AT4G12420 T-DNA insertion Roots skewed and looped away from the normal Sedbrook et al. (2002)
downward direction of growth
SPIRAL 1 (SPR1) AT2G03680 spr1-1/EMS Right-handed helical root growth Nakajima et al. (2004)
spr1-5/T-DNA
insertion
LEFTY 1 ? EMS Left-handed helical growth: epidermal cell files of Thitamadee et al. (2002)
lefty roots begin to skew at the region where first
root hair is emerging
LEFTY 2 ? EMS Left-handed helical growth: epidermal cell files of Thitamadee et al. (2002)
lefty roots begin to skew at the region where first
root hair is emerging
B. Orman et al.
WAVY GROWTH 2 (WAV2) AT5G20520 wav2-1, wav2-2/ Enhanced wavy root growth Mochizuki et al. (2005)
T-DNA insertion
WAG1 AT1G53700 wag1-1, wag1-2/ Wavy root phenotype Santner and Watson
T-DNA insertion (2006)
WAG2 AT3G14370 wag2-1/ T-DNA Wavy root phenotype Santner and Watson
insertion (2006)
AUXIN RESPONSE FACTOR 10 AT2G28350 arf10-2/ T-DNA arf10 arf16 double mutant displays uncontrolled cell Wang et al. (2006)
(ARF10) insertion division and blocked cell differentiation in the
root distal region and shows a tumor-like root
apex and loss of gravity-sensing
AUXIN RESPONSE FACTOR 16 AT4G30080 arf16-2/ T-DNA Uncontrolled cell division and blocked cell Wang et al. (2006)
(ARF16) insertion differentiation in the root distal region. Mutant
shows a tumor-like root apex and loss of gravity-
sensing
ADENYLATE KINASE 2 (AK2) ? T-DNA insertion Mutant exhibits significantly elevated root growth, Carrari et al. (2005),
cap morphogenesis defects, along with alterations Young et al. (2006)
in root sensitivity to gravistimulation and slower
kinetics of root gravitropic curvature
ADENYLATE KINASE 3 (AK3) ? T-DNA insertion Mutant exhibits significantly elevated root growth, Carrari et al. (2005),
cap morphogenesis defects, along with alterations Young et al. (2006)
in root sensitivity to gravistimulation and slower
kinetics of root gravitropic curvature
Elongation zone
QUASIMODO 1 (QUA) AT3G25140 qua1-1, qua1-2/ Reduced cell adhesion. Dwarf phenotype and rough Bouton et al. (2002)
T-DNA insertion aspect resulting from numerous cells protruding
from their cotyledons, leaves, and hypocotyls
PROPORZ 1 (PRZ1) AT4G16420 prz1-1/T-DNA Mutant exhibits defects in cell and organ Sieberer et al. (2003)
insertion differentiation
(continued)
27
28

approach
DAWDLE (DDL) AT3G20550 T-DNA insertion Mutant plants exhibit shortened roots caused by Morris et al. (2006)
lower number of cell divisions
CYTOKININ ROOT SYNDROM ? ckrl-7,-8,-12,-50,- Mutant exhibits significantly elevated root growth Su and Howell (1992)
(CKR1) 09/EMS with shorter root hairs and altered response to
cytokinin
POLARIS (PLS) AT4G39403 Promoter trap Short-root phenotype, relatively short and radially Chilley et al. (2006)
expanded cells, altered response to exogenous
auxins and cytokinins, enhanced ethylene-
response phenotype, defective auxin transport
and homeostasis, and altered microtubule
sensitivity to inhibitors
YADOKARI 1-D (YADK 1-D) ? T-DNA insertion Dwarf mutant: short hypocotyl and primary root, Takase et al. (2004)
reduced apical dominance and reduced number of
lateral roots
MURUS 1 (MUR1) AT3G51160 mur1-1, 1-2/EMS Root growth defects, altered cell walls which are Freshour et al. (2003)
more brittle
BREVIS RADIX (BRX) AT1G31880 T-DNA insertion Roots composed of shorter as well as fewer cells. Mouchel et al. (2004)
Reduction in mature cell size as well as cell
proliferation causes slow primary root growth
PETIT 1 (PT1) ? pet1-1/fast neutrons Defective in aspects of root and hypocotyl elongation Kurata and Yamamoto
and presence of gaps in internal cortical and (1998)
epidermal cell walls
PROCUSTE 1 (PRC1) ? prc1-8, 1-10, 1-12, Decreased cell elongation in roots and dark-grown Fagard et al. (2000)
1-19/ T-DNA hypocotyls
insertion
CONSTITUTIVE EXPRESSION OF AT5G05170 ixr1-1, 1-2/EMS Stunted phenotype. Short hypocotyls in dark-grown Ellis et al. (2002)
VSP1 1 (CEV1) seedlings. Roots have reduced cellulose content,
increased production of jasmonate and ethylene
B. Orman et al.
ARABIDOPSIS THALIANA ACT7 AT5G09810 act7-2, 7-3, 7-4/ Increased root twisting and waving, and retarded root Gilliland et al. (2003)
(ACT7) T-DNA insertion growth. Root apical cells are not in straight files
and contain oblique junctions between cells
WAVE-DAMPENED 2 (WVD2) AT5G28646 Overexpression Constitutive right-handed helical growth in both Yuen et al. (2003)
wvd2-1/Ac/Ds roots and etiolated hypocotyls and impaired
anisotropic expansion.
WVD2-LIKE 1 (WDL1) AT3G04630 Overexpression/ Constitutive right-handed helical growth in both Yuen et al. (2003)
Ac/Ds roots and etiolated hypocotyls and impaired
anisotropic expansion
PICKLE, SUPPRESSOR OF SLR AT2G25170 EMS Primary root differentiates improperly and expresses Li et al. (2005)
2 (PCL) embryonic characteristics after germination
STUNTED PLANT 1 (STP1) ? EMS Roots elongate more slowly than in the WT Baskin et al. (1995),
Beemster and Baskin
(2000)
XYLOGLUCAN AT2G18800 T-DNA insertion Stunted phenotype with shorter root hairs and Liu et al. (2007)
ENDOTRANGLUCOSYLASE/ perturbation in epidermis cell formation
HYDROLASE 21 (XTH21)
ZINC FINGER OF ARABIDOPSIS ? RNAi/ RNAi mediated silencing results in lethality. Devaiah et al. (2007)
THALIANA 6 (ZAT6) overexpression Overexpression affects root development and
retards seedling growth as a result of decreased
Pi acquisition
MULTIDRUG RESISTANCE AT2G47000 pgp4-1, 4-3, 4-4/ Reduced root gravitropic bending and elongation as Terasaka et al. (2005)
P –GLICOPROTEIN (PGP4) T-DNA insertion well as lateral root formation
RESISTANT TO IBA (RIB1) ? Ac/Ds Shorter primary root, increased number of lateral Poupart and Waddell
roots and elongation defects in root gravitropism. (2000), Poupart et al.
Less sensitive to growth inhibition by IBA and (2005)
less sensitive to IBA in stimulation of lateral root
formation
XIPOTL 1 ? T-DNA insertion Short primary root, a high number of lateral roots and Cruz-Ramı́rez et al.
short epidermal cells with aberrant morphology (2004)
and few root hairs

(continued)
29
30

approach
PHOSPHOLIPASE DS 1 (PLDz1) ? T-DNA insertion Slower elongation of primary root and longer lateral Li et al. (2006a, b)
roots in low phosphate conditions
PHOSPHOLIPASE DS 2 (PLDz2) ? T-DNA insertion Slower elongation of primary root and longer lateral Li et al. (2006a, b)
roots in low phosphate conditions
WEAK ETHYLENE AT5G05730 wei2-1, 2-3/EMS Root-specific ethylene insensitivity. Upregulation of Stepanova et al. (2005)
INSENSITIVE 2 (WEI2) WEI2/ASA1 and WEI7/ASB1 by ethylene results
in the accumulation of auxin in the tip of primary
root, whereas loss-of-function mutations in these
genes prevent the ethylene-mediated auxin
increase
WEAK ETHYLENE AT1G25220 wei7-1, 7-2/Ac/Ds Root-specific ethylene insensitivity. Upregulation of Stepanova et al. (2005)
INSENSITIVE 7 (WEI7) WEI2/ASA1 and WEI7/ASB1 by ethylene results
in the accumulation of auxin in the tip of primary
root, whereas loss-of-function mutations in these
genes prevent the ethylene-mediated auxin
increase
HISTONE MONOUBIQUITINATION AT2G44950 hub1-1/EMS hub1-2, Slow primary root growth Fleury et al. (2007)
1 (HUB1) hub1-3/T-DNA
insertion
SCARFACE (SFC) AT5G13300 sfc-9/T-DNA Shorter roots Sieburth et al. (2006)
insertion
Differentiation zone: lateral roots (LR)
ARABIDILLO-1 AT2G44900 T-DNA insertion Fewer lateral roots Coates et al. (2006)
ARABIDILLO-2 AT3G60350 T-DNA insertion Fewer lateral roots Coates et al. (2006)
SUPERROOT 1 (SUR1) AT2G20610 sur1-2, 1-3, 1-4, 1-5, Increased LR number and formation of additional Celenza et al. (1995)
1-6/EMS adventitious root
B. Orman et al.
SUPERROOT 2 (SUR2) AT4G31500 En-1 transposition Numerous adventitious roots begin to grow from the Casimiro et al. (2003),
hypocotyl, lateral root primordial develop at high Casson and Lindsey
frequency, root hairs appear at higher density and (2003)
root elongation is reduced
ABERRANT LATERAL ROOT AT5G11030 alf4-1/gamma rays Unable to produce lateral roots and does not respond Celenza et al. (1995),
FORMATION 4 (ALF4) to exogenous auxins Casimiro et al.
CEGENDUO (CEG) ? T-DNA insertion Increased lateral root production Dong et al. (2006)
KIP-RELATED PROTEIN 2 (KRP2) AT3G50630 Overexpression Mutations do not give any remarkable morphological Himanen et al. (2002)
phenotypes what indicates the presence of
redundant functions. The number of lateral roots
in overexpression line was reduced by 60%
compared with that in the wild type
KANADI (KAN) AT5G16560 kan1-2 /EMS Reduced primary root length and reduced lateral rootHawker and Bowman
(LR) number (2004)
KANADI 2 (KAN2) AT1G32240 kan2-1/EMS Reduced primary root length and LR number Hawker and Bowman
(2004)
KANADI 3 (KAN3) AT4G17695 kan3-1/EMS Reduced primary root length and fewer lateral roots Hawker and Bowman
(2004)
PHABULOSA 6 (PHB6) AT2G34710 EMS Reduced LR number Hawker and Bowman
(2004)
PHAVOLUTA 5 (PHV5) AT1G30490 T-DNA insertion Reduced LR number Hawker and Bowman
(2004)
REVOLUTA 10 (REV10) AT5G60690 T-DNA insertion Reduced LR number Hawker and Bowman
(2004)
AUXIN RESPONSE FACTOR AT5G37020 arf8-1/ T-DNA Long-hypocotyl phenotype in light conditions and Tian et al. (2004)
8 (ARF8) insertion increased formation of LR
AUXIN RESPONSE FACTOR 10 AT1G19220 arf19-1/ T-DNA Mutant with reduced LR development Okushima et al. (2007)
(ARF19) insertion
TRANSPORT INHIBITOR AT3G62980 tir1-9/T-DNA Reduced LR number. Reduced auxin-transport- Xie et al. (2000),
RESPONSE 1 (TIR1) insertion inhibitor-sensitive root elongation Casimiro et al.
(2003)
(continued)
31
32

approach
ENHANCER OF TIR1-1 AUXIN AT1G19220 EMS Auxin-resistant root growth in seedlings and Gray et al. (2003)
RESISTANCE (ETA3) reduced LR development
ARABIDOPSIS SERINE/THREONINE AT1G10940 ask1-1/Ac/Ds Decreased number of LR Fukaki et al. (2005)
KINASE 1 (ASK1)
ASK2 AT3G61160 ask2-1/ T-DNA Decreased number of LR Fukaki et al. (2005)
insertion
CULLIN-ASSOCIATED AND AT2G02560 EMS Decreased number of LR Fukaki et al. (2005)
NEDDYLATION DISSOCIATED,
HEMIVENATA (CAND1)
TRANSPORT INHIBITOR AT3G02260 tir3-1//EMS and Reduced LR number. Reduced auxin-transport- Ruegger et al. (1997),
RESPONSE 3 (TIR3) gamma rays inhibitor-sensitive root elongation Lopez-Bucio et al.
(2005)
ANR1 AT2G14210 ANR1-KO/dSpm Does not show the nitrate-induced stimulatory effect Montiel et al. (2004)
transposon (down-regulated expression)
insertion
ARABIDOPSIS DUAL-AFFINITY ? T-DNA insertion Does not show the nitrate-induced stimulatory effect Zhang et al. (2007)
NITRATE TRANSPORTER GENE
AtNRT1.1 (NRT1.1)
LATERAL ROOT INITIATION (LIN1) AT1G08090 EMS LR development insensitive to high-sucrose, Cerezo et al. (2001),
low-nitrogen medium Casimiro et al.
(2003), Zhang et al.
(2007)
IAA-ALANINE RESISTANT 2 (IAA28/ AT5G25890 iaa28-1/EMS Defective in LR formation, reduced LR number. Lopez-Bucio et al.
IAR2) Defects in root hair development, resistance to (2002)
the stimulatory effects of low P on root hair and
LR formation
MULTIDRUG RESISTANCE- AT1G04120 mrp5-1/T-DNA Increased LR number, decreased root length Gaedeke et al. (2001),
ASSOCIATED PROTEIN 5 insertion Casimiro et al.
(MRP5) (2003)
B. Orman et al.
DWARF IN LIGHT 1 (DFL1) AT5G54510 Activation-tagging Altered hypocotyl length in light and reduced LR Nakazawa et al. (2001),
plasmid number but the primary root length is almost the Casimiro et al.
same as in the WT. Auxin insensitive (2003)
AUXIN RESISTANT 1 (AXR1) AT1G05180 axr1-3/EMS Agravitropic root, reduced LR number. Reduced Lopez-Bucio et al.
auxin-sensitive root elongation (2002), Casimiro
et al. (2003)
AUXIN RESISTANT 2 (AXR2) AT3G23050 axr2-5/T-DNA Agravitropic root, reduced LR number. Short Lopez-Bucio et al.
insertion hypocotyls in dark conditions. (2002)
AUXIN RESISTANT 3 (AXR3) AT1G04250 axr3-3/EMS Reduced root elongation and increased lateral root Lopez-Bucio et al.
number (2002)
AUXIN RESISTANT 4 (AXR4) AT1G54990 axr4-2/gamma rays Reduced LR number. Reduced auxin-sensitive root Lopez-Bucio et al.
elongation (2002), Casimiro
et al. (2003)
AUXIN RESISTANT 5 (AXR5) AT4G14560 axr5-1/unknown Gain-of-function mutation; mutants are resistant to Yang et al. (2004), De
auxin and display a variety of auxin-related Smet et al. (2006)
growth defects including defects in root and shoot
tropisms and reduced LR number on auxin
AUXIN RESISTANT 6 (AXR6) ? atcul1-5/ T-DNA Reduced lateral root number. Reduced auxin- Lopez-Bucio et al.
insertion sensitive root elongation (2002), Casimiro
et al. (2003)
NO APICAL MERISTEM CUP- AT1G56010 RNAi/ RNAi lines have reduced LR number; Montiel et al. (2004),
SHAPED COTYLEDON (NAC1) overexpression overexpressing lines have increased LR number. Scheres et al. (2002),
Xie et al. (2000)
PEROXISOMAL ABC AT4G39850 EMS Reduced IBA-sensitive root elongation. Reduced LR Zolman et al. (2001),
TRANSPORTER 1 (PXA1) number Casimiro et al.
(2003)
SEVEN IN ABSENTIA HOMOLOG 5 AT5G53360 Overexpression Overexpression leads to reduced LR formation Casimiro et al. (2003)
(SINAT5)
PASTICCINO 1 (PAS1) AT3G54010 pas1-1/T-DNA Reduced LR number and short primary root Faure et al. (1998),
insertion Casimiro et al.
(2003)
PASTICCINO 2 (PAS2) AT5G10480 EMS Increased LR number and short primary root Faure et al. (1998)
PASTICCINO 3 (PAS3) ? Reduced LR number and short primary root Faure et al. (1998)
33
(continued)
34

approach
pas3-1, 3-2, 3-3, 3-4/
EMS
SOLITARY ROOT1/ IAA14 (SLR1/ AT4G14550 iaa14-1/ T-DNA Absence of LR development, reduced number of root Scheres et al. (2002),
IAA14) insertion hairs Casimiro et al.
(2003), Montiel et al.
(2004), Fukaki et al.
(2006)
LATERAL ROOT PRIMORDIUM 1 AT5G12330 Gene trap transposon Delayed lateral root initiation Smith and Fedoroff
(LRP1) (1995)
NON-PHOTOTROPHIC AT5G20730 nph4-1, 4-2, 4-3, 4-4/ Decrease in lateral and adventitious root formation Stowe-Evans et al.
HYPOCOTYL (NPH4) fast neutrons (1998), Casimiro
et al. (2003)
SHORT HYPOCOTYL (SHY2) AT1G04240 shy2-2/EMS Significantly shorter roots of shy2-2 mutants with Tian and Reed (1999)
very few lateral roots, whereas the shy2-22 and
shy2-24 mutants form more and much longer LR
than the WT
KNOTTED-LIKE (KNAT6) AT1G23380 T-DNA insertion Down-regulation of KNAT6 expression by RNA Dean et al. (2004),
interference was associated with an increased Montiel et al. (2004)
total number of lateral roots
MASSUGU2/IAA19 (MSG/IAA19) AT3G15540 EMS Defective in lateral root formation and root Tatematsu et al. (2004)
gravitropism
PUCHI ? puchi-1/T-DNA Disturbed cell division patterns in the lateral root Hirota et al. (2007)
insertion primordium, resulting in swelling of the proximal
region of lateral roots
CYTOPLASMIC-INVERTASE 1 ? EMS Insensitivity to osmotic stress-induced inhibition of Qi et al. (2006)
(AtCYT-INV1) lateral root growth. Short primary root, smaller
size of leaves and siliques
B. Orman et al.
LATERAL ORGAN BOUNDARIES- AT2G42430 Overexpression Overexpression induces lateral root formation Okushima et al. (2007)
DOMAIN16 (LBD16)
LATERAL ORGAN BOUNDARIES- AT3G58190 Overexpression Overexpression induces lateral root formation Okushima et al. (2007)
DOMAIN29 (LBD29)
XB3 ORTHOLOG 2 IN ARABIDOPSIS AT5G57740 T-DNA insertion Poor root system and severe defects in lateral root Nodzon et al. (2004)
THALIANA 32 (XBAT32) production. Defective in cell divisions that are
required for lateral root initiation
IAA-LEUCINE RESISTANT 2 (ILR2) AT3G18485 ill2-1/T-DNA Defective in lateral root formation and primary root Magidin et al. (2003)
insertion elongation
IAA-LEUCINE RESISTANT 1 (ILR1) AT3G02875 ilr1-1/EMS Shorter hypocotyl and fewer lateral roots on Rampey et al. (2004)
unsupplemented medium
IAA-LEUCINE-RESISTANT (ILR)- AT5G56660 ill2-1/T-DNA Shorter hypocotyl and fewer lateral roots on Rampey et al. (2004)
LIKE GENE 2 (ILL2) insertion unsupplemented medium
IAA-ALANINE RESISTANT 3 (IAR3) AT1G51760 iar3-1/EMS Shorter hypocotyl and fewer lateral roots on Rampey et al. (2004)
unsupplemented medium
HOMEOBOX-LEUCINE ZIPPER AT4G16780 Overexpression Elevated ATHB-2 levels inhibit specific cell Steindler et al. (1999)
PROTEIN HAT4 (ATHB-2/HAT4) proliferation such as secondary growth of the
vascular system and lateral root formation.
Reduced LR number.
ROOTS CURL IN NP (RCN1) AT1G25490 T-DNA insertion Lateral roots exhibit reduced NPA sensitivity,
gravitropic response and increased auxin
transport.
RHO-RELATED PROTEIN FROM AT1G20090 Overexpression Constitutively active GTP-bound rop2 (CA-rop2): Li et al. (2001)
PLANTS 2 (ROP2) increased LR number; and dominant negative
GDP-bound rop2 (DN-rop2) reduced LR number
SEUSS (SEU) AT1G43850 seu-3/EMS Pleiotropic phenotype that includes reductions in Pfluger and Zambryski
several classic auxin responses such as apical (2004)
dominance, lateral root initiation, sensitivity to
exogenous auxin
ARABIDOPSIS RESPONSE AT1G59940 T-DNA insertion Lateral root formation is more sensitive to cytokinin To et al. (2004)
REGULATOR 3 (ARR3) inhibition

(continued)
35
36

approach
ARABIDOPSIS RESPONSE ? T-DNA insertion Lateral root formation is more sensitive to cytokinin To et al. (2004)
CYTOKININ OXIDASE/ AT2G41510 Overexpression Increased growth of the primary root and increased Werner et al. (2003)
DEHYDROGENASE 1 (CKX1) number of lateral roots
CYTOKININ OXIDASE/ AT5G56970 Overexpression Increased growth of the primary root and increased Werner et al. (2003)
DEHYDROGENASE 3 (CKX3) number of lateral roots
E1-CONJUGATING ENZYME- AT5G19180 ecr1-1/EMS Resistant to the auxin-like compound indole-3- Woodward et al. (2007)
RELATED1-1 (ECR1) propionic acid, produces fewer lateral roots than
wild type, displays reduced adult height
RING-BOX 1 (RBX1) AT5G20570 Overexpression Transgenic plants (35S::RBX1) had smaller Gray et al. (2002)
cotyledons and produced fewer lateral roots than
WT plants
BUSHY AND DWARF (BUD1) AT1G18350 Sense/antisense Significantly fewer lateral roots, loss of apical Dai et al. (2006)
RNA expression dominance, shorter hypocotyl at high temperature
system (29 C) under light. Deficiency in polar auxin
transportt
LONG ? T-DNA insertion Altered hypocotyl length in light and increased LR Casimiro et al. (2003),
HYPOCOTYL 5 (HY5) number. Emergence of LR occurs earlier than in Sibout et al. (2006)
WT, resulting in overall enhanced root system
growth. The gravitropism of hy5 roots is reduced
B. Orman et al.
WALL-ASSOCIATED SER/THR AT1G21210 Antisense gene Impaired cell elongation and blocked LR formation Lally et al. (2001)
KINASE (WAK4)
LIKE AUX1 (LAX3) AT1G77690 T-DNA insertion Nearly 40% reduction in numbers of emerged Swarup et al. (2008)
lateral roots
ABA DEFICIENT 1 (ABA1) AT5G67030 aba1-1/EMS ABA sensitive, reduced ABA inhibitory effect on Signora et al. (2001)
LR length. Shorter primary root
ABA DEFICIENT 2 (ABA2) AT1G52340 aba2-1, 2-3, 2-4/ ABA sensitive, reduced ABA inhibitory effect on Signora et al. (2001)
EMS LR length. Shorter primary root
ABA DEFICIENT 3 (ABA3) AT1G16540 aba3-1/EMS ABA sensitive, reduced ABA inhibitory effect Signora et al. (2001)
3-2/gamma rays on LR length
ABA DEFICIENT 4 (ABA4) ? aba4-1/T-DNA ABA insensitive, reduced ABA inhibitory effect Signora et al. (2001)
insertion on LR length
ABA DEFICIENT 5 (ABA5) ? unknown ABA insensitive, reduced ABA inhibitory effect Signora et al. (2001)
on LR length
ABSCISIC ACID INTENSIVE 1 (ABI1) AT5G57050 EMS Reduced ABA inhibitory effect on LR length De Smet et al. (2003)
ABSCISIC ACID INTENSIVE 1 (ABI2) AT3G24650 EMS Reduced ABA inhibition on LR length De Smet et al. (2003)
ABSCISIC ACID INTENSIVE 1 (ABI3) AT3G24650 EMS Reduced ABA inhibitory effect on LR length De Smet et al. (2003)
ABSCISIC ACID INTENSIVE 1 (ABI4) AT2G40220 Gamma rays Reduced ABA inhibitory effect on LR length De Smet et al. (2003)
ABSCISIC ACID INTENSIVE 1 (ABI5) AT2G36270 abi5-1/T-DNA Reduced ABA inhibitory effect on LR length De Smet et al. (2003)
insertion
ENHANCED RESPONSE TO ABA 1 AT5G40280 unknown Increased number of lateral roots Brady et al. (2003)
(ERA1)
LATERAL ROOT ? EMS Shorter primary root phenotype and ability to Zhang et al. (2007)
ABA-INSENSITIVE (LABI) produce visible LRs in the presence of ABA.
Mutants are less sensitive to the high-nitrate
induced inhibition on LRs
FLOWERING TIME CONTROL AT4G16280 EMS Reduced sensitivity to the inhibitory effect of ABA Zhang et al. (2007)
PROTEIN ALPHA (FCA) on LRs
(continued)
37
38

approach
HOMEODOMAIN-LEICINE ZIPPER AT5G47370 Overexpression Mutations of the HAT2 gene did not produce any Sawa et al. (2002)
PROTEIN HAT2 (HAT2) remarkable morphological phenotypes (mutants
responded to gravity, exogenous auxin, and auxin
transport inhibitor in similar ways to wild-type
plants) indicating the presence of redundant
functions among the HD-Zip II subfamily genes.
35S::HAT2 plants showed reduced lateral root
elongation, and reduced auxin sensitivity
compared to wild-type plants
PI DEFICIENCY RESPONSE ? EMS Disrupted Pi sensing Ticconi et al. (2004)
2 (PDR2)
UBIQUITIN-LIKE MODIFIER AT5G60410 siz1-1, 1-2, 1-3/ Cessation of primary root growth, extensive lateral Miura et al. (2005)
(SUMO) E3 LIGASE (AtSIZ1) T-DNA insertion root and root hair development
WRKY75 AT5G13080 RNAi Suppression of WRKY75 expression through RNAi Devaiah et al. (2007)
silencing results in significantly increased LR
length and number, as well as root hair number
ABERRANT LATERAL ROOT ? alf3-1/EMS Mutant is able to initiate lateral root primordium Celenza et al. (1995),
FORMATION 3 (ALF3) formation but then arrests at the emergence stage Casimiro et al.
(2003)
RELATED TO ABI3/VP1 1 (RAV1) AT1G13260 Overexpression Overexpression causes retarded LR development Hu et al. (2004)
INDOLE-3-BUTYRIC AT2G04550 ibr5-1/EMS Light-grown seedlings have longer primary roots Monroe-Augustus et al.
ACID-RESPONSE (IBR5) with slightly fewer lateral roots. Lateral roots in (2003)
ibr5-1 elongate less than in the WT; hypocotyl
and roots elongate normally in the dark
LRD2 ? EMS Mutant has an altered response to exogenous ABA Deak and Malamy
(2005)
B. Orman et al.
Adventitious roots
ARGONAUTE 1 (AGO1) AT1G48410 EMS Reduced formation of adventitious roots in response Sorin et al. (2005)
to auxin. Defect of hypocotyl elongation in
response to auxin
AUXIN RESPONSE FACTOR 17 AT1G77850 T-DNA/ Overexpression line produces fewer adventitious Sorin et al. (2005)
(ARF17) overexpression roots than the WT
HASTY (HST) AT3G05040 hst-6,/EMS Mutants have reduced size of roots and form Bollman et al. (2003)
hst-7/X-ray adventitious roots from the base of the hypocotyl
ROOT INITIATION DEFECTIVE 1 ? EMS Temperature-sensitive, defective in the initial or the Konishi and Sugiyama
(RID1) pre-morphogenic stage of adventitious root (2003)
formation
formation
formation
formation
ROOT INITIATION DEFECTIVE 5 ? EMS Temperature-sensitive, reduced frequency of root Konishi and Sugiyama
(RID5) initiation at 28 C without affecting the later (2003)
stages of root formation. The rate of adventitious
rooting generally depends on the concentration
of exogenous auxin
ROOT PRIMORDIUM DEFECTIVE 1 ? EMS Temperature-sensitive, mutant can establish Konishi and Sugiyama
(RRD1) adventitious roots but fails to maintain their (2003)
growth. Strongly inhibited subsequent growth of
adventitious roots at 28 C

(continued)
39
40

approach
growth. Strongly inhibited subsequent growth
of adventitious roots at 28 C
growth. Strongly inhibited subsequent growth of
adventitious roots at 28 C
LR lateral root, RAM root apical meristem, QC quiescent centre, WT wild type
B. Orman et al.
the xylem poles (Benjamins and Scheres 2008), whereas in barley from pericycle
and endodermis adjacent to phloem (Briggs 1978) just like in rice and corn
(Hochholdinger and Zimmermann 2008). The general structure of barley lateral
roots seems to be the same as the seminal and nodal roots, despite their different
origins. The transverse section exhibit typical thick-walled endodermis and single
large axile duct surrounded by much more thicker tissue (Gorny 1992). For LR
initiation, auxin plays a crucial role in both monocotyledonous (Chhun et al. 2007)
and dicotyledonous (Tian and Reed 1999; Casimiro et al. 2003) species.
More than 170 genes have been described as important for longitudinal pattern
in Arabidopsis. Alterations in these genes cause often severe phenotype, such as in
the case of GNOM (GN). Mutants of this gene display a range of phenotypes, but all
of them lack a root (Shevell et al. 2000). This gene encodes an ARF GDP/GTP
exchange factor involved in embryonic axis formation and polar localization of
PIN1 (Geldner et al. 2004). It was shown that mutations in this gene disrupt the
polarity of auxin transport and thereby cause defects not only in gravitropism
(Geldner et al. 2003) but also hydrotropism (Miyazawa et al. 2009). Lack of a
primary root is characteristic for BODENLOS (BDL) and MONOPTEROS (MP)
mutants. The MP gene encodes a transcription factor ARF5 (AUXIN RESPONSE
FACTOR 5) that activates auxin-responsive target genes, whereas BDL encodes
INDOLACETIC ACID-INDUCED PROTEIN 12 (IAA12) (Shevell et al. 2000).
Hamann and coworkers (2002) suggested inhibitory effect BDL on MP, but exact
mechanism of their action is unknown (Weijers et al. 2006). Alterations in root
length could be an output of decreased number of cell divisions such as in the case
of DAWDLE (DDL), cell elongation – PHOSPHOLIPASE DS 1,2 (PLDz1) or cell-
wall formation – MURUS 1 (MUR1). DDL mutant plants exhibit shortened roots.
This gene seems to influence transcription activation by recruiting proteins to
transcription complexes; however, its precise function is still unknown (Morris
et al. 2006). Slower elongation of primary roots and faster of lateral roots in low
phosphate conditions are characteristic for PLDz1 and PLDz2 mutants. These genes
are involved in root elongation during phosphate limitation – they promote primary
root growth but inhibit lateral root elongation (Li et al. 2006b). MUR1 mutants
exhibit root grow defects, where more brittle altered cell walls are observed. This
gene is necessary to form essential pectin cross-links within the cell wall and proper
composition of cell wall polysaccharides (Freshour et al. 2003).
Up to now, many genes have been described as involved in lateral root formation
in the differentiation zone. Lateral roots are formed from the pericycle “founder
cells,” which undergo a series of periclinal and anticlinal divisions to generate a
new meristem (Casson and Lindsey 2003). One of the earliest genes involved in
lateral root formation is ALF4 (ABERRANT LATERAL ROOT FORMATION 4).
The ALF4 mutant is unable to produce lateral roots or adventitious roots and
does not respond to exogenous auxins (Casimiro et al. 2003). It was suggested by
DiDonato and coworkers (2004) that ALF4 functions in maintaining the pericycle
in the mitotically competent state needed for lateral root formation. There are only
few mutants described as involved in lateral root emergence. LAX3, which has been
described recently by Swarup et al. (2008), encodes an auxin influx carrier that
42 B. Orman et al.
facilitates emergence of new primordia. Mutants exhibit nearly 40% reduction in

numbers of emerged lateral roots. Many genes involved in lateral meristem activa-
tion are related to ABA, such as ABA DEFICIENT 1 (ABA1). This mutant has
shorter primary root, is ABA-sensitive, and exhibit reduced ABA inhibition of LRs
length (Signora et al. 2001). As auxin is involved in all steps of lateral root
formation, genes involved in ABA metabolism determine auxin-independent
checkpoint for lateral root development. The product of the ABA1 gene – zeaxan-
thin epoxidase – generates the epoxycarotenoid precursor of the ABA biosynthetic
pathway (Barrero et al. 2005).
Little is known about adventitious root formation in Arabidopsis. Among those
genes, ARGONAUTE 1 (AGO1) has been well described. Mutants are barely able to
form adventitious roots in response to auxin and exhibit defect of hypocotyl
elongation in response to auxin. Sorin et al. (2005) suggested that AGO1 regulates
genes required for adventitious root development through its action on the regula-
tion of ARF17 expression. Mutation in AGO1 results in the higher levels of ARF17
expression in hypocotyl, which in turn leads to fewer adventitious roots. ARF17-
overexpressing line also forms fewer adventitious roots than the wild type (Sorin
et al. 2005).
2.3 Root Mutants in Monocotyledonous Species Published

in Pubmed
The deepest monocotyledonous root system is usually of seminal origin, whereas

the upper layers of the soil are penetrated by the nodal roots (Gorny 1992). In
addition to their white color, nodal roots are much thicker and less branched than
seminals and maintain larger number of root hairs. The anatomy of nodal roots
differs from seminal roots. Young ones have all thin-walled stele cells. There are
several (four to six) large ducts in the center surrounded by parenchymatous cells.
Moreover, the xylem and phloem are undetectable. Eight to nine layers of paren-
chymatous cells form the cortex separated from the stele by the endodermis. The
fully developed roots exhibit four large ducts separated by the more thick-wall
cells. Each of twelve to sixteen xylem groups contains one large vessel. The groups
are separated from each other by parenchyma cells and phloem poles hard to
distinguish. Outside the endodermis, there are six to eight layers of large parenchy-
matous cortical cells (Jackson 1922).
Up to now, little is known about genes involved in root architecture in mono-
cotyledons. The main information came from three species: rice, maize, and wheat
(Table 2.3). Similar to dicotyledons, also forward and reverse approaches were used
to study root traits. At least six mutants were obtained trough Mu transposition, four
by g-irradiation, three by NaN3, and one by each: MNU, Tos17, and tissue culture.
Reverse approach (e.g., RNAi, overexpression) were also used to study influence of
a gene of interest on root traits. Several mutants have been described, which are
responsible for monocotyledonous root traits. Lim and coworkers (2005) described
Table 2.3 Mutated genes responsible for monocotyledonous plants root architecture
Gene name (alias) Accession Mutation Mutant phenotype References
approach
Root traits
RAN-RELATED GTP-BINDING PROTEIN AF488730 Overexpression Increased primordial tissue, reduced number Wang et al. (2006)
(TaRAN1) of lateral roots and stimulated
hypersensitivity to exogenous auxin
SCARECROW (ZmSCR) AF263457 EST-based Not reported Lim et al. (2005)
isolation
OsASR1 ? ? Defective seminal roots Ge et al. (2004)
CROWN ROOTLESS 1 (ZmCRLL1) BG873644 ? Not reported Inukai et al. (2005)
CROWN ROOTLESS 2 (ZmCRLL2) BE050765 ? Not reported Inukai et al. (2005)
CROWN ROOTLESS 1 (ZmCRL1) AY736375/ MNU Impaired initiation of nodal root primordia Liu et al. (2005), Inukai
AB200234 et al. (2005)
CASEIN KINASE 1 (OsCKL1) AJ487966 Antisense Reduced primary root length and fewer lateral Liu et al. (2003)
and nodal roots
CROWN ROOTLESS 1 (OsCRLL1) AB200235 ? Not reported Inukai et al. (2005)
CROWN ROOTLESS 2 (OsCRLL2) AB200236 ? Not reported Inukai et al. (2005)
CROWN ROOTLESS 3 (CRLL3) AB200237 ? Not reported Inukai et al. (2005)
CROWN ROOTLESS 4 (CRLL4) AB200238 ? Not reported Inukai et al. (2005)
GLUTAMATE RECEPTOR LIKE DQ305408 T-DNA insertion Short root phenotype Li et al. (2006a, b)
CHANNEL 3.1 (GLR3.1)
GLUCOSAMINE-6-PHOSPHATE AY772189 T-DNA insertion Short root phenotype Jiang et al. (2005)
ACETYLTRANSFERASE (GNA1)
ROOT ARCHITECTURE ASSOCIATED 1 AY659938 Overexpression More nodal roots, shorter primary and lateral Ge et al. (2004)
(OsRAA1) roots compare to WT
ROOTHAIR DEFECTIVE 3 (TaRHD3) AY557340 mRNA Not reported Shan et al. (2005)
differential
display
OsCRL2 ? Gamma rays Impaired initiation and growth of nodal root Inukai et al. (2001)
primordia, longer primary root than WT
43
(continued)
44
Gene name (alias) Accession Mutation Mutant phenotype References

approach
ROOTLESS WITH UNDETECTABLE ? Mu insertion
Deficient in the initiation of the embryonic Woll et al. (2005)
MERISTEMS 1 (RUM1) seminal roots and the postembryonic
lateral roots on the primary root
ALTERED LATERAL ROOT FORMATION ? Retrotransposon Significantly shorter lateral roots as compared Rani Debi et al. (2003)
(OsALF) Tos17 with the wild type
REDUCED ROOT LENGTH 1 (OsRRL1) ? Gamma rays Reduced root length Inukai et al. (2001)
REDUCED ROOT LENGTH 2 (OsRRL2) ? Gamma rays Reduced root length Inukai et al. (2001)
SHORT LATERAL ROOTS 1 (ZmSLR1) ? Mu insertion Short lateral roots as a result of impaired root Hochholdinger et al.
cell elongation (2001)
SHORT LATERAL ROOTS 2 (ZmSLR2) ? Mu insertion Short lateral roots as a result of impaired root Hochholdinger et al.
cell elongation (2001)
SHORT LATERAL ROOTS 5 (OsSRT5) ? NaN3 Reduced root length Yao et al. (2003)
SHORT LATERAL ROOTS 6 (OsSRT6) ? NaN3 Reduced primary root length and diameter, the Yao et al. (2003)
mutant at the seedling stage also shows
inhibited lateral root elongation and altered
root hair formation
PIN-FORMED 1 (PIN1) NM_001063762 RNAi Significantly inhibited nodal root emergence Xu et al. (2005)
and development
PINOID (PID) NM_001073800 Overexpression Delay of nodal root development, curled Morita and Kyozuka
growth of shoots and agravitropic roots (2007)
SLENDER (SLR1-1) XM_469478 Gamma rays Reduced number and root length compared Ikeda et al. (2001)
with the wild-type plant
ADP-RIBOSYLATION FACTOR (ARF) NM_001062427 Overexpression Reduced apical dominance, shorter primary Zhuang et al. (2005)
GTPASE-ACTIVATING PROTEIN roots, increased number of longer nodal
(GAP) (AGAP) roots.
AUXIN EFFLUX MUTANT (AEM1) ? ? Short lateral roots, reduced development of Rani Debi et al. (2005)
root hairs, agravitropic root
ADVENTITIOUS ROOTLESS 1 (OsARL1) ? Tissue culture Defective in nodal root formation Liu et al. (2005)
B. Orman et al.
Root hairs
CELLULOSE SYNTHASE-LIKE D1 BK000089 Ds gene trap Root hair development is initiated normally, Kim et al. (2007)
(OsCSLD1) the hairs elongate less than the wild-type
hairs and they have kinks and swellings
along their length
ROOTHAIRLESS 3 (ZmRTH3) ? Mu insertion Impaired in root hair elongation Wen and Schnable (1994)
ROOT HAIRS (OsRH2) ? NaN3 Defective in the formation of root hairs Ma et al. (2001)
ROOTHAIRLESS 1 (ZmRTH1) AY265854 Mu insertion Impaired in root hair elongation Wen and Schnable (1994),
Wen et al. (2005)
ROOTHAIRLESS 3 (ZmRTH3) AY265855 Mu insertion Impaired in root hair elongation Wen and Schnable (1994),
Hochholdinger et al.
(2008)
NaN3 sodium azide, WT wild type
45
46 B. Orman et al.
maize ZmSCR gene. They suggested that this gene is Arabidopsis SCR ortholog
based on sequence and expression pattern similarity to the members of the GRAS
family. It was then confirmed due to the ability to complement the Arabidopsis SCR
mutant phenotype, which suggests conservation of function. Although the main
knowledge about lateral root development came from Arabidopsis, rice mutant
ALF1 (ALTERED LATERAL ROOT FORMATION) has been isolated by Rani Debi
and coworkers (2003). This mutant displayed not only significantly shorter lateral
roots as compared with wild type but also reduction in both the number and length
of root hairs. In maize, SHORT LATERAL ROOTS1 (SLR1) and SLR2 mutants have
been reported with defective lateral root elongation (Hochholdinger et al. 2001).
The defects in both mutants act specifically during early postembryonic root
development, and crown roots at all the stages produced normal lateral roots similar
to the wild type. In contrast, the ALF1 mutant displays shorter lateral roots in both
embryonic seminal and postembryonic crown roots up to later growth stages (Rani
Debi and coworkers, 2003). Rice mutants that lack CELLULOSE SYNTHASE-LIKE
D1 (OsCSLD1) function develop abnormal root hairs that elongate less. It appears
that OsCSLD1 may be the functional ortholog of Arabidopsis KOJAK, which is
involved in root hair elongation (Kim et al. 2007). The similar phenotype is
observed in maize roothairless 3 (ZmRTH3), which encodes a COBRA-like protein
(Hochholdinger et al. 2008).
2.4 Strategy for EST Data-Mining
The goal of this work was to find an optimal, short, and efficient procedure in a search
for potential orthologs between Arabidopsis and barley using rice for confirmation
and between already reported genes in other monocotyledons and barley. The first
step was to review the literature in searching for genes that are described as involved
in root development. Out of 259 Arabidopsis and 35 monocotyledonous genes found
in this search, it was possible to analyze a total number of 192 Arabidopsis and
21 monocotyledonous genes, whose nucleotide and protein sequences were available
in GenBank database. Potential orthologs between Arabidopsis and barley and
between other monocotyledons and barley were analyzed separately.
2.4.1 Searching for Potential Orthologs Between Arabidopsis

and Barley
The strategy included two pipelines (Fig. 2.1). First, a search in the GeneBank for
rice potential orthologs using BLASTn and BLASTp based on Arabidopsis nucleo-
tide and protein sequences, respectively, was done. To minimize false positive
results, more restrictive criteria (E value 105 or less) were chosen than suggested
Searching in databases for publications on root

mutants in Arabidopsis
(PubMed in GenBank)
259 Arabidopsis genes
Nucleotide sequence
Protein sequence Searching in TIGR for similar sequences (ESTs) in
Identification of gene sequences responsible for
barley, wheat and maize, potential homologs of
mutant phenotype in Arabidopsis
Arabidopsis
(PubMed in GenBank)
(BLASTn)
192 Arabidopsis genes
Nucleotide sequence
Nucleotide
Protein sequence
sequence
Confirmation
Searching in GenBank for similar sequences in rice Using ETS sequences from barley to search for
genome similar sequences in rice
(BLASTn and BLASTp) The same rice (BLASTx and BLASTp)
sequences found in
both searches 50 Arabidopsis genes
Arabidopsis nucleotide
and protein sequence
Comparision of Arabidopsis, rice and barley Comparision of exon/intron arrangement between
Barley nucleotide sequences to each other on nucleotide and protein Arabidopsis and rice
and protein sequences level (EMBL ClustalW) (Jellyfish)
CDD search (BLASTp)
Rice nucleotide
and protein sequences
Fig. 2.1 Strategy for selection of potential barley orthologs to Arabidopsis genes. E value
(GenBank)/Expect (TIGR) 105 or less
by Pevsner (2003). However, it should be noticed that BLAST is a heuristic version

of Smith-Waterman algorithm, so it generates an output that is a list of sequences
based on Score value obtained for each corresponding fragment (Koonin and
Galperin 2004). In other words, the more points the alignment gets, the higher on
the output list will the sequence be. Moreover, change of the parameters of BLAST
searching modifies the Score value for each alignment and may automatically have
an influence on the order of sequences in the result list. That is the main reason for
the need to manually verify the results from BLAST searches using multiple
alignment tool ClustalW.
Parallel to this, the search for barley ESTs in TIGR and GenBank databases was
performed to select Arabidopsis genes, which have good EST coverage. To mini-
mize false positive results, more restrictive criteria were chosen (Expect 105 or
less) just like in the previous searches. The barley EST sequences were then used as
a query in TIGR database in search for rice ESTs. Rice ESTs obtained through this
searching were then aligned with rice nucleotide and protein sequences obtained
through GenBank searching.
Using this approach, 22 genes involved in LR formation, 19 genes controlling
root development, and 8 genes involved in root hair formation in Arabidopsis
(which lead to total number of 49 genes) were identified (Table 2.4). To determine
the level of similarity between Arabidopsis, barley, and rice, the sequences were
compared on nucleotide and protein level. Nevertheless, the success of this
approach depends heavily on the quality of EST sequences, which cannot be
guaranteed. This is mostly due to the existence in EST artifacts during cDNA
library construction and inherent errors caused by DNA sequencing procedures
Table 2.4 Arabidopsis genes which have potential orthologs in barley and rice genome
48
Arabidopsis Rice Barley Similarity [%] References

Alias Gene acc. no. Alias Gene acc. no. EST(s) acc. no. At-rice At-barley
N P N P
Lateral root development
ABA1 At5g67030 ? Os04g0448900 TC159565 60.6 57.1 69.6 59.8 Signora et al. (2001)
TC189509 70.2 61.5
AUX1 AT2G38120 LAX2 Os01g0856500 TC177829 51.6 79.9 70.5 86.4 Fukaki et al. (2005)
Os05g0417200 32.7 8.84
AXR1 At1g05180 ? Os03g0820100 TC168107 60 66.1 66.3 66.4 Lopez-Bucio et al. (2002)
AXR2 At3g23050 IAA30 Os12g0601300 TC183661 49.4 55.8 62.6 83.1 Lopez-Bucio et al. (2002)
IAA13 Os03g0742900 TC182343 59.6 53.1
CKX1 AT2G41510 ? Os01g0940000 TC153934 53.6 55 63 64.7 Werner et al. (2003)
Os05g0374000 11.6 4.69
CKX3 At5g56970 ? Os05g0374200 TC153934 51.6 40.1 56.5 46.3 Werner et al. (2003)
Os01g0197700 39.9 27.1
Os01g0187600 28.7 17.7
Os01g0775400 53.1 45.1
LIN1 At1g08090 ? Os02g0112600 TC163374 60.5 69.9 68.3 72 Casimiro et al. (2003), Zhang et al. (2007)
RCN1 AT1G25490 ? Os09g0249700 TC163839 66.5 83.5 75.0 81.8 Rashotte et al. (2001)
TC174461 74.7 82.6
SINAT5 AT5G53360 SINAT5 Os05g0238200 TC156156 53.8 53.9 45 82.9 Xie et al. (2002), Casimiro et al. (2003)
Os02g0293400 48.2 56.5
SLR1 AT4G14550 IAA14 Os12g0601300 TC182343 56.7 53.0 75.5 79.5 Casimiro et al. (2003)
Os03g0742900 58.2 53.1
TIR1 AT3G62980 TIR1 Os05g0150500 TC168970 48.9 57.7 64.8 64.0 Casimiro et al. (2003)
WRKY75 AT3G01970 WRKY75 Os11g0490900 TC185610 46.1 37.1 74.5 78.4 Devaiah et al. (2007)
PHV AT1G30490 ATHB14 Os03g0640800 TC176589 62.0 72.8 69.5 71.4 Hawker and Bowman (2004)
HOX33 Os12g0612700 67.5 69.6
CAND1 AT2G02560 TIP120 Os02g0167700 TC192976 65.4 76.0 69.7 72.6 Cheng et al. (2004)
PAS2 AT5G10480 ? Os01g0150200 TC166551 56.8 75.5 70.4 71.6 Faure et al. (1998)
ILL2 AT5G56660 ILL1 Os01g0706900 TC173699 51.4 51.6 67.6 62.5 Rampey et al. (2004)
B. Orman et al.
IAR3 AT1G51760 ILL1 Os01g0560000 TC173699 57.1 63.3 67.0 68.0 Magidin et al. (2003)
TC158327 63.8 63.5
ECR1 AT5G19180 ? Os01g0271500 TC165467 60.5 67.8 71.1 78.7 Woodward et al. (2007)
TC172176 68.7 69.8
RBX1 AT5G20570 RBX1a Os01g0106800 TC161421 59.1 80.1 77.0 85.0 Gray et al. (2002)
REV AT5G60690 HOX9 Os10g0480200 TC177627 68.6 70.4 63.3 62.2 Hawker and Bowman (2004)
HOX10 Os03g0109400 TC176589 68 69 70.6 70.7
TC182801 72.7 80.9
AGL21 AT4G37940 MAD21 Os02g0579600 TC187224 65.3 60.1 56.2 39.4 Zhang et al. (2007)
SUR1 AT2G20610 ? Os11g0552000 TC164962 53.4 46 59.2 50.7 Casimiro et al. (2003)
Root hair development
ERH3 AT1G80350 KATANIN Os01g0683100 TC160566 63.3 81.0 71.3 76.6 Schneider et al. (1997)
p60
RHL2 AT5G02820 RHL2 Os03g0284800 TC174707 56.8 61.4 67.8 63.4 Schneider et al. (1997)
RHD1 AT1G64440 GEPI48 Os05g0595100 TC192755 60.8 74.2 61.4 57.4 Schiefelbein and Somerville (1990)
GEPI48 Os08g0374800 60.6 62
TIP1 AT5G20350 TIP1 Os02g0184000 BU996187 58.7 63.8 52.1 57.3 Hemsley et al. (2005)
TIP1 Os06g0644500 EH091151 64.5 64.3 44.5 40.2
TC179087 42.9 46.1
RHD2 AT5G51060 RBOHD Os11g33120 TC153922 42.2 47.7 56.0 63.8 Schiefelbein and Somerville (1990)
Os12g0541300 55.7 49.5 58.4 54.7
67.5 66.4
KJK AT3G03050 CSLD2 Os06g0111800 TC164787 64.4 79.8 73.7 85.8 Favery et al. (2001)
CSLD1 Os10g0578200 TC187976 59 75.5 64.9 64.3
65.5 60.7
59.2 54.4
RHD3 AT3G13870 RHD3 Os01g0575000 TC168229 64.0 62.4 69.0 70.1 Schiefelbein and Somerville (1990)
TC181503 73.8 82.9
TC169425 66.8 66.5
TC177006 65.2 63.7
ROP2 AT1G20090 RAC6 Os02g0120800 TC179704 56.4 88.3 78.3 89.3 Li et al. (2001)
(continued)
49
50
Arabidopsis Rice Barley Similarity [%] References

Alias Gene acc. no. Alias Gene acc. no. EST(s) acc. no. At-rice At-barley
N P N P
Primary root architecture
SMT1 AT5G13710 SMT1 Os07g0206700 TC155984 63.4 77.3 71.5 77.0 Willemsen et al. (2003)
Os03g59290 66.1 65.2
Os11g19140 45.7 32.
RML1/ AT4G23100 GSH1B Os05g0129000 TC162201 57.6 65.3 72.4 78.8 Vernoux et al. (2000)
GSH1 Os07g0462000 TC194971 62.4 61.2 74.3 80.2
HPA AT1G71920 HPA Os02g0709200 TC155290 60.7 68.9 69.2 76.8 Mo et al. (2006)
DDL1 AT3G20550 SNIP1 Os05g0545600 TC161796 36.3 23.7 70.3 69.7 Morris et al. (2006)
TC158236 62.4 58.2
CEV1/ AT5G05170 CESA8 Os07g0208500 TC187976 66.2 79.5 76.0 84.9 Ellis et al. (2002)
CESA3 CESA2 Os03g0808100 72.7 69.8 61.9 50.9
59.9 39.8
AtXTH21 AT2G18800 ? Os06g0697000 TC166673 46.5 53.4 63.4 59.1 Liu et al. (2007)
PAS1 AT3G54010 PAS1A Os03g0367000 TC162239 63.4 64.2 72.8 71.7 Casimiro et al. (2003)
70.6 64.0
TC158273 70.6 64.0
COB AT5G60920 COBL3 Os05g0386800 TC170213 48.1 69.9 67.9 66.1 Scheres et al. (2002)
COBL4 Os03g0754500 TC165216 45.5 44.5 66.7 37.5
COBL1 Os10g0497700 m 59.9 54.2
COBL2 Os07g0604300 62.8 64.5
Os03g0416300 64.9 62.6
KEULE AT1G12360 SEC1A Os04g0252400 TC190473 36.6 56.6 61.2 58.9 S€
29.1 68.3 65.7
70.1 70.8
74.6 80.3
Os02g0452500 TC161457 31.8 29.1 68.3 65.7
TC184788 70.1 70.8
B. Orman et al.
TC174383 74.6 80.3

KNOLLE AT1G08560 ? Os03g0736500 TC179685 58.1 56.8 56.8 45.8 S€
Os12g08980 44.6 25.9
IRX1 AT4G18780 CESA4 Os01g0750300 TC160053 60.6 73.4 70.3 61.7 Taylor et al. (2000)
75.2 82.6
72.7 77.7
IRX3 AT5G17420 CESA9 Os09g0422500 TC154161 66.7 80.6 70.0 76.2 Taylor et al. (2000)
66.6 72.1
64.8 67.0
HLR At4g29040 ? Os03g0298400 TC156203 68.9 95.1 76.9 93.6 Ueda et al. (2004)
SKU 5 AT4G12420 ? Os10g0508000 TC158012 57.5 62.4 65.6 53.5 Sedbrook et al. (2002)
Os06g0104300 BG300903 64.3 67.5 61.4 66.4
WAV2 AT5G20520 MAD18 Os07g0608300 TC166812 57.3 69.3 69.9 74.1 Mochizuki et al. (2005)
KOR1 AT5G49720 GUN9 Os03g0329500 TC154162 68.4 74.6 67.4 72.1 Zuo et al. (2000)
GUN10 Os03g0736300 56.4 59
RCE1 AT4G36800 ? Os08g0374100 TC161238 43.9 30.4 69.3 48.2 Larsen and Cancel (2004)
Os09g0321900 41.4 28.2
Os10g0190000 43.6 20.1
FASS AT5G18580 TONNEAU2 Os05g0149800 TC172751 73.9 85.1 76.8 88.5 Torres-Ruiz and J€ urgens (1994)
MUR1 AT3G51160 GMD2 Os06g0137700 TC162999 62.3 70.2 70 79.3 Freshour et al. (2003)
Acc. no accession number, At Arabidopsis thaliana, N nucleotide, P protein
Bold indicates the most probable ortholog
51
52 B. Orman et al.
(Liang et al. 2007), because ESTs are single pass reads. This leads to comparison of
only corresponding fragments of sequences to determine similarity. Moreover,
ESTs may often provide information on only a partial segment of an entire
cDNA, whereas random sampling of clones leads to redundancy in EST datasets,
as mention by Parkinson et al. (2002). To minimize false negative results in
generation of barley consensus sequences, the CAP3 program was used, which
has an ability to clip 50 and 30 low quality regions of reads (Huang and Madan
1999). To prevent “domain hits” (e.g., similarities that are caused by the conserva-
tion of fragments within families), only these Arabidopsis/monocotyledons
sequences were chosen, which have extended barley EST coverage beyond the
domain zone. Each time, the domain area on a nucleotide sequence, based on CDD
search using Jellyfish, was established manually. As previously suggested by
McGinnis and Madden (2004), the fastest way to compare the function of a protein
is to perform a CDD search, which uses a database of motifs to characterize
“conserved-domains” in a protein sequence. Following this idea, each selected
sequence, which led to the confirmation of the existence of the same conserved
domain in all cases (data not shown), was submitted into such analysis.
2.4.2 Arabidopsis and Rice Genes Comparisons
The definition of gene homology implies the existence of a common ancestor gene,
which existed before speciation (in the case of orthologs) or before duplication (in
relation to paralogs) (Alexeyenko et al. 2006). This implies the conservation in
exon/intron arrangement between homologous genes, which led to the comparison
of exon/intron organization in selected Arabidopsis and rice genes. In most cases,
the arrangement was highly conserved between putative homologs, whereas some
of them exhibited deletions or insertions (Fig. 2.2). Nevertheless, these changes
have not disturbed an overall order in exon/intron arrangement.
2.4.3 Searching for Potential Orthologs Between Other

Monocotyledons and Barley
Due to the lack of genomic sequences for most of monocotyledonous genes, it was
not possible to check the level of conservation of exon/intron arrangement. Just like
in the previous case, the first step was to search for barley ESTs in TIGR and
GenBank databases (Fig. 2.3). This allowed selection of monocotyledonous genes,
which have good EST coverage in barley genome, following the rules described
above. Parallel to this, searching was done for the rice (in case of maize and wheat
genes) and Arabidopsis sequences in GenBank. The barley ESTs were then used in
a search for rice ESTs, which were compared with rice sequences from GenBank.
As mentioned above, this step was performed to establish whether these sequences
RCN1 (ROOTS CURL IN NP)

A. thaliana SINAT5 (SEVEN IN ABSENTIA HOMOLOG 5)
A. thaliana
83,3% 53,9%
O. sativa O. sativa
1000 bp 1000 bp
AGL21 (AGAMOUS-LIKE 21) A. thaliana
ECR1 (E1-CONJUGATING ENZYME-RELATED1-1)
A. thaliana
60%
68%
O. sativa O. sativa
1000 bp
1000 bp
PAS1 (PASTICCINO 1)
A. thaliana
PHV5 (PHAVOLUTA 5) A. thaliana
64%
71%
O. sativa
1000bp
1000 bp
Level of similarity
10–0 % 30–20 % 50–40 % 60–70 % 80–90 %
20–10 % 40–30 % 60–50 % 70–80 % 90–100 %
Fig. 2.2 Examples of exon/intron arrangement in othologous Arabidopsis and rice genes.
corresponding fragments are shaded using appropriate color in response to similarity between
these fragments on protein level; black line ¼ scale bar
are the same to confirm that the “hit” did not occur only by chance. This analysis led
to the total number of ten genes, including six rice, two maize, and two wheat genes,
which have potential orthologs in barley genome (Tables 2.5 and 2.6). ClustalW
was also used for determining the similarity between other monocotyledons and
barley sequences on nucleotide and protein level, respectively. To establish poten-
tial domains of barley proteins, CDD search was performed and confirmed in all
cases the existence of the same conserved domains as in monocotyledonous
proteins (data not shown).
2.4.4 Phylogenetic Analysis
Even if the pairwise approach was theoretically the most powerful one-to-one
methodology to predict true orthologs, many phylogenetic methods have been
well described up to now (Chiu et al. 2006; Hulsen et al. 2006; Conte et al.
2008). In order to confirm the output from manually created BLAST-based
approach and to establish the relationships between each of Arabidopsis and rice
genes, it was decided to use GreenPhyl pipeline, which has been described as the
54 B. Orman et al.
Searching in databases for publications on root

mutants in monocotyledonous spiecies
(PubMed in GenBank)
35 monocotyledonous
genes
Nucleotide sequence
Identification of gene sequences responsible for Protein sequence Searching in TIGR for similar sequences (ESTs) in
mutant phenotype barley, wheat and maize, potential homologs of
(PubMed in GenBank) monocotyledonous genes (BLASTn)
21 monocotyledonous
genes
Nucleotide sequence
Nucleotide Protein sequence
sequence
Searching in GenBank for similar sequences in Confirmation Using barley ETS sequences to search for similar
Arabidopsis and rice genomes (in case of maize and sequences in rice
wheat genes) (BLASTx and BLASTp)
(BLASTn and BLASTp) The same rice sequences found
in both searches 11 monocotyledonous
genes
Arabidopsis nucleotide
and protein sequence
Comparision of Arabidopsis, rice and barley
Barley nucleotide sequences to each other on nucleotide and protein
and protein sequences level (EMBL ClustalW)
CDD search (BLASTp)
Rice nucleotide
and protein sequences
Fig. 2.3 Strategy for selection of potential barley orthologs to monocotyledonous genes. E value
(GenBank)/Expect (TIGR) 105 or less
most efficient phylogenetic method (Conte et al. 2008). In many cases, a large
number of proteins showing high sequence similarity to Arabidopsis were encoded
in the rice genome (data not shown). This is likely to be the result of multiple rounds
of gene and genome duplications, followed by differential gene loss (Adams and
Wendel 2005; Sterck et al. 2007). Following Conte et al. (2008), only ortholog
associations in which a bootstrap value was 50% and more were taken into account
as statistically significant. The total number of 50 Arabidopsis and 11 monocotyle-
donous genes were analyzed using this approach. From this number, 26 Arabidopsis
genes (13 genes involved in LR formation, 3 genes involved in root hair formation,
and 13 genes involved in root development) were confirmed as potential orthologs
with a bootstrap value 50% or more (Fig. 2.4). Only in case of three Arabidopsis
and one monocotyledonous genes, the orthologs detected by GreenPhyl were
different from these selected on the basis of BLAST searching. Although genes
selected as potential orthologs using BLAST approach were on the phylogenetic
tree, they had lower bootstrap value. For genes typed by phylogenetic approach, the
GreenPhyl bootstrap values were higher than values for genes selected using
BLAST and were above 50%.
2.4.5 Synteny Detection in Arabidopsis and Rice Genomes
To establish whether gene orders remained conserved between Arabidopsis and

rice putative orthologs, the “Cinteny” pipeline was used (Sinha and Meller 2007).
From 50 Arabidopsis sequences selected as having potential orthologs in rice
Table 2.5 Monocotyledonous genes which have potential orthologs in barley and Arabidopsis genomes
Monocotyledonous Rice Arabidopsis Barley Similarity [%] References
species
Alias Gene acc. Alias Gene acc. no. Alias Gene acc. EST(s) acc. Mc-Os Mc-Hv At-Os
no. no. no. N P N P N P
Ta RAN1 AF488730 RAN1B Os05g0574500 RAN3 AT5G55190 TC176612 75.9 98.1 94.3 100 81.3 94.5 Wang et al. (2006)
Zm RTH1 AY265854* RTH1 Os03g0625700 – AT1G47550 TC160970 56.5 65.6 86.8 92.6 24.9 25.8 Wen et al. (2005)
At1g47560 TC187549 24.7 25.3
Zm RTH3 AY265855 COBL7 Os03g0301200 COBL7 AT4G16120 TC157935 61.9 86.7 82.2 58.5 57.7 50 Hochholdinger et al.
COBL8 At3g16860 57.6 48.2 (2008)
COBL9 At5g49270 56 49.2
Ta RHD3 AY557340 RHD3 Os01g0575000 RHD3 AT3G13870 TC168229 79.6 90.1 97.2 97.4 68.9 69.7 Shan et al. (2005)
AT1G72960 TC177006 95.4 96.8 64.1 63.8
55
56
Table 2.6 Rice genes which have potential orthologs in barley and Arabidopsis genomes
Rice Arabidopsis Barley Similarity References
Os-At Os-Hv
Alias Gene acc. no. Alias Gene acc. no. EST(s) acc. no. N P N P
Os CRL1 Os03g05510 JLO AT4G00220 NP9937331 46.6 36.6 64 41.8 Inukai et al. (2005)
Os CKL1 Os02g0622100 CKI1 AT4G14340 TC176778 49.4 66.8 86.5 92 Liu et al. (2003)
CKL10 At3g23340 67 66.6
Os CSLD1 Os10g0578200 KJK AT3G03050 TC164787 59 75.5 72.4 78.7 Kim et al. (2007)
TC157331 80.7 –
Os RAA1 Os01g0257300 FLP1 At4g31380 BM816685 66.5 58.9 75.0 78.1 Ge et al. (2004)
FPF1 AT5G24860 62.8 58.9
At5g10625 41.5 39.7
Os PIN1 Os02g0743400 PIN1 At1g73590 TC188592 57.1 68.3 86.5 53.4 Xu et al. (2005)
TC164300 82.9 82.1
Os SLR1-1 XM_469478 GAI At1g14920 TC156386 44.6 53.9 84.8 85.0 Ikeda et al. (2001)
RGA1 At2g01570 45.7 53.1
RGL1 At1g66350 46.5 51.4
RGL3 At5g17490 48.2 49.4
RGL2 At3g03450 44.5 53.1
Os AGAP Os05g0489600 ATARFA1B AT5G14670 TC166447 51.7 60.6 83.3 75.7 Zhuang et al. (2005)
AT1G70490 61.7 59.5
At1g10630 60.6 59.5
AT3g62290 61.6 29.5
At2g47170 59.5 59.5
Acc. no.accession number, At Arabidopsis thaliana, Os Oryza sativa, Hv Hordeum vulgare, N nucleotide, P protein
Bold indicates the most probable ortholog
B. Orman et al.
Fig. 2.4 The bird’s eye on in silico analysis: best candidates for molecular cloning. Using
GreenPhyl, potential ortholog associations in barley genome were considered to be significant if
the supporting bootstrap value was 50% and more. Similarity searching was proceeded using E
value (GenBank)/Expect (TIGR) 105 or less. Genes that are situated in the middle (belonging to
all three wheels) represent genes that have been selected by smart “best hit” strategy using BLAST
searching and obtained a phylogenetical confirmation using GreenPhyl (bootstrap value 50% and
more), and the conservation of gene order has been confirmed by Cinteny. Genes that are listed in
the BLAST wheel were selected based on “best hit” strategy and have a GreenPhyl bootstrap value
lower than 50%. GreenPhyl wheel corresponds to those genes that have candidates with bootstrap
value higher than 50%, while “best hit” approach selected other candidate genes that have lower
bootstrap values. Those genes, which belong to Cinteny wheel, preserved conservation in gene
order. Genes that belong to BLAST and GreenPhyl wheels were selected by “best hit” approach
and have bootstrap value 50% and more, but Cinteny did not display synteny blocks and/or
orthologs in Arabidopsis or rice genome. Genes that belong to both GreenPhyl and Cinteny and
separately to BLAST and Cinteny exhibit conservation of gene order for genes that belong to
GreenPhyl and BLAST wheels, respectively
genome, 34 exhibited conservation in gene order (15 genes involved in LR forma-

tion, 6 genes involved in root hair formation, and 12 genes involved in root
development). For the rest of 16 Arabidopsis genes, orthologs were not detected
in rice genome using synteny-based approach. Nevertheless, it has been shown
previously that, where microcolinearity is broken, it is possible to find “missing”
gene in nonorthologous locus (Xu et al. 2002; Ware and Stein 2003). That is the
reason why the lack of synteny does not imply the absence of homology. On the
58 B. Orman et al.
other hand, the conservation of gene order during evolution could be treated as a
valuable confirmation.
2.5 In Silico vs. Laboratory Approach to Gene Identification
Information from model species could be used in gene identification in two general
ways. The first one is based on laboratory approach, where designing of degenerate
starters (Ma et al. 1990; Finnegan and Dennis 1993) or probes for screening libraries
(Schmidt et al. 1993; Nomura et al. 2003) have been commonly used. The second
one is a bioinformatic approach, which in most cases is based on sequence similarity
search using BLAST, phylogenetical analysis (Conte et al. 2008), as well as on the
existence of synteny, as suggested by Fritz-Laylin and coworkers (2005). In general,
the combined strategy is commonly used, which is based on bioinformatic analysis
followed by molecular verification, like suggested in this paper.
In spite of their obvious successes in the past, laboratory strategies alone are
inappropriate for large-scale analysis. The main disadvantage is their pure
sequence-based nature, which can generate false-positive results, especially in
correspondence to evolutionary divergence, where the level of similarity based on
sequence comparison could be very low.
The improvements in sequencing technology led to hundreds of complete
genome sequences, though most come from microorganisms. Till the end of
2008, only the genomes of three dicotyledonous species (A. thaliana, Populus
trichocharpa and Vitis vinifera), one monocotyledonous species (O. sativa), and a
moss (Physcometrilla patens) have been fully sequenced. Recently also, complete
draft assembly of the soybean (Glycine max) and maize (Zea mays) were released.
Although, new sequencing technologies are now available, the assembly of large
and complex genomes is still hampered by a significant content of repetitive DNA
and, in allopolyploids, by the presence of homoeologous genomes. Most
of economically important crops, specifically bread (16,979 Mbp) and durum
(12,030 Mbp) wheat, barley (5,100 Mbp), oat (12,961 Mbp), rye (7,933 Mbp),
and maize (2,793 Mbp), have large genomes (Doležel et al. 2007). For most of
them, deep collections of full-length cDNA sequences are not available. In silico
methods that are based on phylogenomic analysis suffer because of the lack of
universal and efficient method for generating phylogenetic trees (Fu and Jiang
2007). Even the full genomic sequence does not guarantee the propriety of such
analysis. It has to be taken into account that this could straightly lead to mistakes
because of wrongly generated phylogenetic tree, as suggested by Dutilh et al.
(2007). However, before the start of the genome sequencing projects, large-scale
EST-sequencing projects were undertaken in several cereal species, and a large
number of ESTs have become available for most of them. In spite of their impor-
tance (Varshney et al. 2006; Liang et al. 2007), EST projects yielded mostly partial
cDNA sequences, which are not adequate for direct comparison and assembly of
entire genes. Nevertheless, the increasing amount of ESTs unlocks the gene con-
tents of many species and automatically creates a need to elaborate new strategies to
use this knowledge. They could be analyzed using only sequnce-based approach,
like BLAST or FASTA, but such strategy can generate mistakes (Koonin and
Galperin 2004).
Here is proposed the EST-based combined procedure for selecting potential
orthologs, which is based on BLAST analysis combined with phylogenetic- and
synteny-based approaches. The strategy includes a simple searching procedure used
as a confirmation, which can avoid most common pitfalls during BLAST exploita-
tion. Moreover, manual verification of the position of the evolutionary conserved
fragments of proteins in domain zones using CDD search and Jellyfish program
minimizes the risk of the so-called “domain hits,” especially when the protein
family is large. Although it should be noticed that lack of synteny does not imply
absence of homology, such searching can be very handful during selection of genes.
It was demonstrated in the presented paper that bioinformatic analysis is a powerful
tool, which gives the possibility to find potentially homologous sequences between
two species. The procedure that combines three most commonly used in silico
approaches allowed to shortlist the number of potential orthologs as good candi-
dates for molecular cloning.
2.6 Methods
2.6.1 Rice and Arabidopsis Searches
Searches for rice and Arabidopsis genes were carried out in publicly available
genome databases. Arabidopsis sequences were obtained from The Arabidopsis
Information Resource (TAIR) database (http://www.arabidopsis.org/). O. sativa
sequences being potential homologs of A. thaliana genes were chosen using
mRNA and protein sequences of A. thaliana genes searched against the GenBank
database using BLASTn and BLASTp with default parameters, respectively (http://
blast.ncbi.nlm.nih.gov/Blast.cgi). Among a large number of output sequences
obtained from the search, we selected the potential orthologs based on carefully
selected criteria. First, E value was very restrictive and lower than 105 (Pevsner
2003). Each of the searches has been done in both directions to avoid hits obtained
just “by chance.” These sequences were also identified as potential orthologs
through phylogenetic analysis using GreenPhyl (http://greenphyl.cines.fr/cgi-bin/
greenphyl.cgi) or OrthologID; alternatively (http://nypg.bio.nyu.edu/orthologid/)
synteny detection was proven using Cinteny (http://cinteny.cchmc.org/).
60 B. Orman et al.
2.6.2 Sequence Analysis
The next stage of bioinformatic analysis was to check the degree of similarity
on protein level between A. thaliana and O. sativa. The putative O. sativa and
A. thaliana orthologous genomic sequences retrieved were then aligned with
mRNA sequences for intron/exon junction positions, respectively, using Jellyfish
program (http://jellyfish.labvelocity.com). This application was also used to align
exon(s) of A. thaliana to the corresponding ones in O. sativa on protein level.
Alignments of protein sequences were performed at The European Molecular
Biology Laboratory (http://www.ebi.ac.uk/embl/ ) using the CLUSTALW program
(Chenna et al. 2003) with default parameters.
2.6.3 ESTs
Searches for ESTs used in the presented publication were performed in publicly
available EST libraries in The TIGR Gene Indices (Quackenbush et al. 2001) using
the BLASTn and tBLASTx program with default parameters (http://www.tigr.org/
db.shtml). This includes: barley sequences release 10.0 (June 3, 2008), wheat
release 11.0 (July 13, 2008), maize release 18.0 (July 18, 2008), and rice release
17.0 (June 20, 2006). Searches for barley EST sequences corresponding to chosen
monocotyledonous and Arabidopsis genes were also made in the GenBank EST
database (http://www.ncbi.nlm.nih.gov/dbEST/index.html) using the tblastn pro-
gram and default parameters.
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Chapter 3
Genomics of Root–Microbe Interactions
Ulrike Mathesius and Giel E. van Noorden
Contents
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
3.2 Genomics Resources for Studying Root–Microbe Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . 75
3.2.1 Legume Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
3.2.2 Microorganism Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.3 Insights into Root–Microbe Interactions Using Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.3.1 Initial Communication Between Roots and Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.3.2 Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.3.3 Root Endosymbiosis, Endoparasitism, and the Regulation
of Defense Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
3.3.4 Alteration of Root Development by Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.3.5 Nutrient Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.3.6 Feedback Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
3.4 Conclusions and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.1 Introduction
All plants coinhabit their environment with a multitude of microorganisms, includ-

ing bacteria, viruses, fungi, nematodes, and protozoans. In many cases, plants
interact with specific microbes, leading to symbiotic relationships, where both
partners are intimately associated and can either mutually benefit, or one partner
can live at the other’s expense. Roots are in close contact with the soil and an array
of microorganisms that inhabit the rhizosphere. Easily available carbon is usually in
short supply in soils, and microorganisms can benefit from root exudates and dead
root material as a food source. Sometimes they specifically invade living root tissues
to access nutrients from the plant. In the case of pathogenic interactions, this may
lead to damage or death of the plant tissue. Common root-pathogen relationships
U. Mathesius (*) and G.E. van Noorden

Australian Research Council Centre of Excellence for Integrative Legume Research, Division of
Plant Science, Research School of Biology, Australian National University, Linnaeus Way,
Canberra, ACT 0200, Australia
e-mail: Ulrike.Mathesius@anu.edu.au; Giel.Vannoorden@anu.edu.au

74 U. Mathesius and G.E. van Noorden
include interaction of roots with pathogenic root knot (Meloidogyne sp.) or cyst
(Heterodera and Globodera sp.) nematodes, infection of roots by pathogenic fungi,
oomycetes, or bacteria. In contrast, plants and microbes have also evolved important
mutualistic symbioses, most notably the interaction of plants with nitrogen-fixing
bacteria and with mycorrhizal fungi. In both cases, the invading microbial partner
provides nutrients in the form of ammonia (nitrogen-fixing bacteria) or phosphorus
(mycorrhizal fungi), in exchange for carbon sources from the plant.
Because of the economic importance of the latter two mutualistic interactions, a
major research effort has focused on unraveling the molecular basis of these
symbioses. One of the best studied interactions is that between legumes and
nitrogen-fixing soil bacteria called rhizobia. Rhizobia invade the roots of specific
legume partners through root hairs or via crack entry, largely avoiding plant
defense responses. Rhizobia produce species-specific lipochitin oligosaccharides
(Nod factors) which are perceived by plant LysM-like receptors and activate a
signal transduction pathway required for the invasion process and the subsequent
development of a new root organ, the nodule (Geurts et al. 2005; Riely et al. 2004).
Rhizobia remain outside the plant cytoplasm and are engulfed in a symbiosome
membrane, which functions to regulate nutrient exchange between the partners.
Nodules arise from redifferentiating root pericycle and cortical cells and are later
invaded by rhizobia (Hirsch 1992). After further growth and differentiation of the
nodule, the rhizobia start converting nitrogen from the air into ammonia, which is
exported to the plant as amino acids. In exchange, rhizobia import carbon from the
plant. This nutrient exchange requires coordination of transport processes by both
partners (Prell and Poole 2006). The Rhizobium-legume (hereafter abbreviated RL)
symbiosis also requires feedback mechanisms, so that symbiosis can be limited at
times of sufficient nitrogen supply of the plant (Caetano-Anollès and Bauer 1988).
In contrast to the limited host range of rhizobia on legumes, most land plants
form a mutualistic symbiosis with mycorrhizal fungi. Fungal hyphae show
increased hyphal branching in the vicinity of host roots and invade root tissues,
forming either arbuscular structures inside root cortical cells (arbuscular mycor-
rhizae or AM) or extracellular hyphal structures (ectomycorrhizae or EM). In AM
symbioses, which are the most widespread associations and have existed for the last
450 M years, fungal hyphae first colonize the root surface where they form
appressoria, invade roots intercellularly through clefts formed by the plant partner
between epidermal cells, followed by intracellular invasion of root cortical cells
and the formation of arbuscules in the inner root cortex (Harrison 2005). Similar to
rhizobia, the fungal partner remains separated from the plant cytoplasm by a
perifungal membrane. There is intensive nutrient exchange across membrane inter-
faces between the fungus and the plant. The most important nutrient provided by
the AM fungal partner is phosphorus, while the plant provides carbon and lipid
sources for the fungal symbionts (Harrison 1999). Again, feedback regulation
functions to limit the carbon supply of the plant to the symbiont, which has been
estimated to reach 30% of the total plant assimilated carbon (Nehls et al. 2007).
Root endoparasitic nematodes can cause enormous losses to crop plant production
and have thus been extensively studied. The most common of these nematodes
3 Genomics of Root–Microbe Interactions 75
include root knot and cyst nematodes, obligate sedentary endoparasites that complete
their life cycle within the roots of host plants. Both invade roots and form feeding
structures into which they divert large amounts of plant nutrients, leading to plant
deformation or death (Bird and Koltai 2000; Williamson and Gleason 2003). The
mechanism of gall or cyst formation is not well understood, but most likely a result of
injections from nematode glands. Root knot nematodes induce giant cells, resulting
from acytokinetic mitosis (mitosis without cell division) and endoreduplication of
xylem parenchyma cells, which is accompanied by cell proliferation in cortical and
pericycle cells, leading to root gall formation (Goverse et al. 2000a). Cyst nematodes
induce the formation of syncytia, multinucleate cells resulting from fusions of cell
contents of multiple root cells as well as endoreduplication of those cells (Goverse
et al. 2000a). Both feeding structures alter transfer of nutrients from the xylem into
the feeding site in a one-way relationship, in contrast to mutualistic symbionts.
Studying root–microbe interactions has provided insight into a number of
biological processes, for example, recognition and communication of partner organ-
isms (Cooper 2007), elicitation and suppression of defense responses (Samac and
Graham 2007), formation and maintenance of endosymbiotic structures (Kistner
and Parniske 2002), remodeling of plant development and meristem activity by the
microbial partner (Ferguson and Mathesius 2003), nutrient exchange (Benedito et al.
2006), and long distance signaling in the plant (Beveridge et al. 2007). We will focus
our review on aspects of these processes after discussing some of the major model
organisms and genomic tools available for studies into root– microbe interactions.
3.2 Genomics Resources for Studying Root–Microbe

Interactions
3.2.1 Legume Resources
As neither the RL nor AM symbioses are formed in Arabidopsis, model legumes have
been in the forefront of genomics research into root–microbe interactions. The
selection of Medicago truncatula and Lotus japonicus as model plants for the study
of RL and AM symbioses by a large community of researchers greatly contributed to
the amount resources that are available for genomic approaches (Cook 1999; Udvardi
et al. 2005). Both legumes have small diploid genomes of 470–550 Mb in size, have
short regeneration times, are self-fertile, and are relatively easy to transform and
regenerate. Both M. truncatula and L. japonicus are currently targets of genome
sequencing projects, which have helped significantly in the map-based cloning of
genes required for root–microbe interactions. As of January 2007, 176 Mb of nonre-
dundant sequences of the L. japonicus and 189 Mb of nonredundant sequences of the
M. truncatula genomes have been released. These correspond to approximately 40%
of the entire genome of both legumes and cover 69 and 58% of public expressed
sequence tags (ESTs) of L. japonicus and M. truncatula, respectively (Sato et al.
2007). The crop legume soybean (Glycine max) has been proposed as a third model
legume and sequencing is well underway (Jackson et al. 2006; Stacey et al. 2004).
Soybean is a model legume for other bean species with more complex genomes and
has been extensively studied for its interactions with rhizobia and cyst nematodes.
In addition to the genome sequencing projects, large EST databases are available
for legumes that have been useful for transcript analyses as a basis for protein
identification in proteomics studies and for the development of transcript profiling
arrays (Journet et al. 2002). EST frequency analyses (in silico Northers) have also
been used for transcript profiling (Tesfaye et al. 2006). For M. truncatula, around
200,000 ESTs are available (MtDB2 http://www.medicago.org/MtDB/; http://
compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb¼medicago). ESTs from
L. japonicus are available from Kazusa at http://est.kazusa.or.jp/en/plant/lotus/
EST/ and from Harvard University at http://compbio.dfci.harvard.edu/tgi/cgibin/
tgi/gimain.pl? gudb¼l_japonicus. EST sequences from soybean are numerous
(330,436) and can be found at http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/
gimain.pl?gudb¼soybean.
For M. truncatula, both a 16k microarray and The Affymetrix GeneChip1
Medicago Genome Array are available. The 16K microarray (Medicago truncatula
Mt16kOLI1 70mer oligonucleotide-based microarray) is based on all tentative
consensus sequences (TCs) from the DFCI Medicago gene index release 5.0
(Hohnjec et al. 2005). The Affymetrix GeneChip® Medicago Genome contains
about 48,000 transcripts of M. truncatula, 1,850 transcripts of M. sativa (alfalfa),
and all the genes of Sinorhizobium meliloti, the symbiont of M. truncatula and
M. sativa. An Affymetrix chip is also available for soybean and includes over
37,500 soybean transcripts as well as 15,800 transcripts for Phytophthora sojae (an
oomycete pathogen of soybean) as well as 7,500 transcripts from the soybean cyst
nematode (Heterodera glycines). Genomics resources for L. japonicus include
cDNA arrays and Serial Analysis of Gene Expression (SAGE) (Sato et al. 2007).
In addition, suppressive subtractive hybridization (SSH) has been used in a number
of studies to identify transcripts differentially displayed in specific cDNA libraries.
Proteomics is another postgenomic tool that has gained steadily in popularity
and has been used in several root–microbe studies (Bestel-Corre et al. 2004). For
both M. truncatula and L. japonicus, protocols for proteomic analysis are available
in protocol handbooks (see below), although much development is needed for
detection of low abundance proteins, phosphoproteins, and other posttranslational
modifications.
Metabolic profiling is a third postgenomic tool and is the most complex in scope
and so far limited in its use. To measure metabolites on a genomics scale requires
specialized equipment such as high-performance liquid chromatography, capillary
electrophoresis, and gas chromatography in combination with mass spectrometry.
In addition, the metabolite profiling data are highly complex, which presents
challenges for identification and quantification of the metabolites. Metabolomics
was used to study metabolite profiles in mature nodules in L. japonicus (Desbrosses
et al. 2005) and M. truncatula (Barsch et al. 2006), as well as in mycorrhizal roots
(Schliemann et al. 2008). Carbon, nitrogen, and phenylpropanoid metabolism have
been the major focus of published metabolomic studies. In addition, a metabolic
pathway database has been established (Urbanczyk-Wochniak and Sumner 2007).

A major current limitation is the availability of chemical reference databases for
identification of a larger number of metabolites.
Research into the biology of root symbiosis in these model legumes is supported
by a range of postgenomic resources (Ané et al. 2008; Colebatch et al. 2002b, c)
including reverse genetic approaches as gene silencing by RNA interference
(RNAi), virus-induced gene silencing, T-DNA and transposon tagging, fast neutron
and EMS (ethyl methanesulfonate) mutagenesis (Tadege et al. 2005), and bioinfor-
matics resources (Cannon et al. 2005; K€ uster et al. 2007a; Lamblin et al. 2003).
A TILLING (Targeting Induced Local Lesions IN Genomes) service has been set
up for both legumes at the John Innes Centre, Norwich, UK. Further descriptions of
these resources and detailed protocols for the study of root–microbe interactions in
these species can be found in the handbooks for M. truncatula (http://www.noble.
orgMedicagoHandbook/) and L. japonicus (Marquez et al. 2005).
3.2.2 Microorganism Resources
In recent years, several Rhizobium strains have been sequenced (MacLean et al. 2007)
including Sinorhizobium meliloti, the symbiont of M. truncatula (Galibert et al. 2001),
Mesorhizobium loti, the symbiont of L. japonicus (Kaneko et al. 2000), and Bradyr-
hizobium japonicum, the symbiont of soybean (Kaneko et al. 2002). The complete
sequenced genomes of these rhizobia allowed many genomic studies including
profiles of transcript (Perret et al. 1999) and protein expression (Djordjevic et al.
2003; Djordjevic 2004). The AM fungus Glomus intraradices and the EM fungus
Laccaria bicolour are two symbiotic fungal genomes being sequenced (http://darwin.
nmsu.edu/~fungi/index.php). Sequencing projects for several root knot and cyst
nematodes (http://www.nematode.net) and for root pathogenic fungi and oomycetes
(http://www.broad.mit.edu/annotation/fgi/, http://genome.jgi-psf.org/) are underway.
Recent reviews give an update on genomics of fungal partners (Soanes et al. 2002,
2007), discuss studies on transcript profiling during host–pathogen interactions, and
give an excellent overview on design of these experiments (Wise et al. 2007). We will
therefore not cover these areas in our chapter in detail.
3.3 Insights into Root–Microbe Interactions Using Genomics
3.3.1 Initial Communication Between Roots and Microbes
The first step in root microbial interactions is mutual recognition and subsequent
attraction of the microbe to the root surface. Following signal molecule recognition,
signal transduction is necessary to initiate defense reactions, morphological
changes, or physiological adaptations of the root and whole plant.
Both plants and microbes release chemical signals into the rhizosphere that aid
in mutual recognition and attraction. Legumes have long been known to release
species-specific mixtures of (iso) flavonoids into the soil, which are recognized by a
number of organisms. Rhizobia perceive flavonoids of their host legumes by
binding of the flavonoid to a protein called NodD, which then activates a suite of
nodulation genes inside the bacteria (Redmond et al. 1986). This gene induction by
flavonoids appears specific to nodulation-related genes: a proteome analysis of
Rhizobium leguminosarum in response to flavonoids revealed only four altered
proteins (Guerreiro et al. 1997), and a transcriptome study of S. meliloti showed
only nine altered gene transcripts (Capela et al. 2005). The requirement for root
flavonoids for the successful induction of Nod genes and subsequent nodulation has
recently been shown in soybean, where silencing of the isoflavonoid pathway by
RNAi led to an inhibition of nodulation, which could be overcome by inoculating
plants with a flavonoid hypersensitive Bradyrhizobium strain or purified Nod
factors (Subramanian et al. 2006). Flavonoids of certain structures are also active
as stimulators for mycorrhizal fungi and can trigger hyphal growth and branching
that can be observed before AM fungi infect the root (Steinkellner et al. 2007).
However, the successful infection of plants defective in flavonoid synthesis has cast
doubt on a strict requirement for flavonoids for the AM symbiosis (Becard et al.
1995). Strigolactones are a class of sesquiterpenoid compounds that are released
from roots of mycorrhizal host, but not from nonhost plants, and have been the first
identified compounds with activity as stimulators of hyphal branching in AM fungi
(Akiyama et al. 2005).
Microorganisms in turn produce a range of signaling molecules that mediate
root–microbe interactions and have extensive effects on the host. The best studied
of these signals are the Nod factors synthesized by rhizobia. Nod factors are
necessary for nodulation and sufficient for the early signaling events in the root.
Nod factors not only induce specific nodulation-related responses but also have
effects on root growth and lateral root formation (Olah et al. 2005). A large-scale
SSH approach identified many new regulatory genes activated in roots following
the first 48 h after Nod factor treatment (Godiard et al. 2007).
Most bacteria release so-called “quorum sensing” signals (QSS) that are used in
communication between bacteria and the regulation of a range of bacterial behaviors
that require coordination between bacterial cells, including pathogenic behaviors of
rhizosphere bacteria (von Bodman et al. 2003). While several studies have shown the
extent of gene expression changes in the bacteria in response to QSS (Arevalo-Ferro
et al. 2003; Chen et al. 2003; Gao et al. 2007; Schuster et al. 2003), it has become
apparent that plant hosts can also detect and broadly respond to QSS. A proteome
analysis of M. truncatula roots showed over 100 protein changes in response to QSS,
and these were specific for the QSS structure and concentration (Mathesius et al.
2003). In addition, treatment of roots with QSS led to changes in the expression of
disease-related genes in the shoots of tomato plants, indicating that QSSs have
systemic effects in the plant that alter plant defense (Schuhegger et al. 2006).
Therefore, it is likely that eukaryotes have evolved detection systems for signals
that could alert plants to the presence and density of bacterial symbionts of pathogens
in the rhizosphere (Bauer and Mathesius 2004).
The existence of a mycorrhizal factor (“Myc factor”) has long been suggested.
Firm evidence for a diffusible factor from AM fungi comes from experiments,
where expression of a plant reporter gene, ENOD11::gusA, was activated in
response to AM fungi that were physically separated from the root by a membrane
(Kosuta et al. 2003). Interestingly, the diffusible factor still stimulated ENOD11
gene expression in a dmi (does not make infections) mutant that is unable to form
either nodules or arbuscules, suggesting that the “Myc factor” triggers early signal
transduction pathways outside this essential signal cascade. So far the “Myc factor”
has not been structurally identified.
A recent study has suggested the existence of a “Nem factor,” a signaling
molecule released by parasitic root knot nematodes (Weerasinghe et al. 2005).
This signal is likely to act on the same signal transduction pathway as Nod factors,
as the nematode signal was unable to initiate root responses in mutant host plants
lacking a functional Nod factor receptor (Weerasinghe et al. 2005). Identification of
the “Myc” and “Nem” factors would be an important advance, together with
characterization of genes involved in their synthesis and regulation, which is
expected to progress with the sequencing of fungal and nematode genomes (Bird
et al. 2005; McCarter et al. 2005).
3.3.2 Signal Transduction
Unraveling of the signal transduction pathways required for successful microbial

invasion and symbiosis has been accelerated in recent years through the positional
and mapped-based cloning of key genes of the signal transduction pathways,
especially in RL and AM symbioses. Importantly, Nod factor receptor candidates,
as well as a calcium signaling cascade and several crucial transcription factors,
were identified. An interesting finding of those studies was that there is a group of
early signal transduction genes in legumes that are required for both RL and AM
symbioses. Several detailed recent reviews have covered the identification and
characterization of these genes (Cook 2004; Gianinazzi-Pearson and Brechenmacher
2004; Harrison 2005; Kinkema et al. 2006; Oldroyd and Downie 2006; Parniske
2004; Stacey et al. 2006), and therefore these studies will not be discussed in detail
here. The identification of one of the signal transduction genes, the calcium-
calmodulin dependent kinase, DMI3, has been one of the first examples of transcript-
based cloning (Mitra et al. 2004a), whereby a transcript profiling comparison of the
mutant and wild type was used to identify a few candidate genes with changed
expression, including the mutant gene.
The nodulation mutants are now being used increasingly as tools in postgenomic
analyses to study the downstream effects these mutations have on root–microbe
interactions. For example, a transcriptome analysis found that gene expression
changes induced in wild-type roots in response to rhizobia were not activated in
six early nodulation-deficient (nod) mutants and only partially induced in a later
nod mutant (hcl, hair curling). In addition, it was shown that the responses of 46
selected genes were specifically due to Nod factor synthesis by the rhizobia (Mitra
et al. 2004b). Similar results were found in a micro- and macroarray analysis of M.
truncatula that identified more than 750 gene differentially displayed during the
first 10 days of nodule development (El Yahyaoui et al. 2004). Expression changes
can be detected within 1 h of inoculation with rhizobia and showed stage-specific
patterns (Lohar et al. 2006).
In the AM symbiosis, the dmi3 mutant, which does not form AM or RL
symbioses, fails to regulate several genes altered by AM in the wild type, including
a receptor kinase, transcription factors, an ABC transporter, and an auxin response
gene (Sanchez et al. 2005). Similarly, several other genes were only induced by AM
in wild type but not the dmi3 mutant, and interestingly, these genes could be induced
even in absence of physical contact between fungi, suggesting that a diffusible
“Myc” factor triggers the responses (Weidmann et al. 2004). In addition, an extensin
and a Nod-like gene with similarity to membrane proteins showed reduced induc-
tion in the dmi3 mutant at the appressorium stage and this might be linked to cell
wall modifications necessary for the infection structure (Siciliano et al. 2007).
A study of gene expression changes in response to AM fungi in seven early signal
transduction mutants in L. japonicus that are affected in AM-colonization identified
several gene expression changes dependent on the mutations (Kistner et al. 2005).
Additional components of signal transduction pathways that are shared and
specific to the RL and AM symbiosis were identified in gene expression profiles,
including a large number of transcription factors and kinases, but their roles remain
to be investigated (Deguchi et al. 2007; Frenzel et al. 2005; Hohnjec et al. 2005,
2006; Liu et al. 2003; Manthey et al. 2004). A combination of in silico and
transcript profiling has highlighted (AM and RL)-symbiosis-specific genes and
promoter elements in M. truncatula, as reviewed by K€uster et al. (2007b). Since
the finding that most of the early signal transduction genes are required for both AM
and RL symbioses, it has been interesting to search for genes specific for each
symbiosis. Of interest are a group of lectin-like genes that are specifically induced
during AM and RL symbioses and could play a role in binding cell wall carbohy-
drates of the microsymbiont and recognition of the partners (Frenzel et al. 2005;
Mitra and Long 2004). A large (>300) group of short proteins with a signaling
peptide and a cysteine motif has been identified to be specific for nodules in
indeterminate legumes (Mergaert et al. 2003). This was confirmed and extended
by in silico studies searching for nodule-specific genes (Fedorova et al. 2002;
Tesfaye et al. 2006). Comparative transcript profiling of AM- and RL-infected
roots showed AM-specific expression of two putative transcription factors that
could be involved in gibberellic acid (GA) signaling (Manthey et al. 2004).
Interestingly, comparisons of AM-induced genes with those induced in interaction
of roots with the pathogenic fungi Magnaporthe grisea and Fusarium moniliforme
(Guimil et al. 2005) and with the growth promoting bacterium Pseudomonas
fluorescens (Sanchez et al. 2005) showed large overlaps in the root’s response to
these very different microbes, suggesting similarities in their perception.
3.3.3 Root Endosymbiosis, Endoparasitism, and the Regulation

of Defense Responses
The successful invasion of microbes into plant roots requires physical changes in
the root, formation of infection structures, and the regulation of defense responses,
so that the invading microbe is tolerated by the root and restricted to certain tissues.
In many legumes, rhizobia infect roots through infection threads (ITs) that form in
infected root hairs. Other legumes are infected at so-called crack-entry sites at
lateral root bases and these differences might reflect evolutionary stages in nodula-
tion (Sprent 2007). The aquatic legume Sesbania rostrata can be infected in both
ways, depending on growth conditions. When flooded, ethylene build-up inhibits IT
entry and rhizobia invade by crack entry. A transcriptome comparison of S. rostrata
roots infected via IT and crack entry identified multiple transcripts specific for each
process (Capoen et al. 2007). A calcium-dependent protein kinase (CDPK1) was
shown to be necessary for effective infection by rhizobia (and AM fungi) and
transcript analysis of roots in which CDPK1 was silenced showed altered expres-
sion of cell wall and defense-related proteins (Ivashuta et al. 2005).
It has been suggested that rhizobia inhibit plant defense responses for successful
invasion (Mith€ ofer 2002). In recent years, transcriptomics and proteomics studies
found evidence for large-scale changes in root defense responses. Transcript
profiling of early stages of nodulation showed that the majority of defense-related
transcripts was induced early (from 1 h) after inoculation but was repressed during
later stages, especially during IT development (Lohar et al. 2006). In nodules, there
is evidence for enhanced expression of defense-related genes, and this might reflect
the ongoing control of the bacterial partner by the plant (Colebatch et al. 2002a,
2004; El Yahyaoui et al. 2004; Tesfaye et al. 2006). Ethylene is one of the hormones
mediating defense responses. The notion that nodulation is restricted by abortion of
infection events by the plant was supported by the hyperinfection and hypernodula-
tion of an ethylene insensitive mutant (sickle) (Penmetsa and Cook 1997). This
mutant shows an altered expression of putative defense-related proteins, for exam-
ple Kunitz proteinase inhibitor, trypsin inhibitor, and a pathogen-related protein
(Prayitno et al. 2006a). Salicylic acid (SA) and jasmonic acid (JA) also play a role
in regulating defense responses, and there is evidence that Nod factors down-
regulate defense responses mediated by SA (Martinez-Abarca et al. 1998) and
that JA biosynthesis is enhanced during the early stages of infection (Kouchi
et al. 2004).
In AM roots, fungal hyphae are restricted to cortical cell layers, and defense
responses are likely to limit hyphal spread. Several studies have used transcrip-
tomics and proteomics to identify candidates that play a role in defense and disease
resistance. Successful infection by AM fungi appears to be related to a week early
but transient expression of defense-related genes (or often just a downregulation
without induction), followed by later induction of defense gene expression in
arbuscule-containing cells, similar to the RL symbiosis (Deguchi et al. 2007;
Garcia-Garrido and Ocampo 2002; Gianinazzi-Pearson and Brechenmacher 2004;

Liu et al. 2003).
The AM- and RL-deficient dmi3 mutant of M. truncatula showed induction of a
disease resistance gene during early appressorium formation in the AM symbiosis,
suggesting that DMI3 might be involved in early downregulation of defense
responses as part of successful invasion (Siciliano et al. 2007). Amiour et al.
(2006) showed in a proteomic study that several glutathione-S-transferases (GST)
are downregulated in appressorium-forming roots, which could play a role in
defense. In contrast, SSH studies have shown increased abundance of GSTs in
AM-infected roots and suggested that in addition to defense, this gene might be
involved in arbuscule senescence (Brechenmacher et al. 2004; Wulf et al. 2003).
In addition to local gene expression changes, mycorrhizal fungi were also shown to
induce systemic changes in the shoot that led to increased pathogen resistance in
M. truncatula, accompanied by expression changes of defense- and stress-related
genes (Liu et al. 2007). The latter study also showed that most of the induced genes
are common between roots inoculated with three different species of mycorrhizal
fungi (G. intraradices, G. versiforme, and Gigaspora gigantea). Similar findings
were made in M. truncatula inoculated with a range of different AM fungi that
induced largely similar responses (Massoumou et al. 2007). Defense-related
changes to gene expression found in many studies by genomic techniques could
explain well known observations that AM-infected roots are more resistant to
pathogen attack (Cordier et al. 1998; Liu et al. 2007) and might become important
targets in improving plant health.
The extent of defense responses appears to be affected by the combination of
host and fungal partner. A study by Feddermann et al. (2008) differentiated
responses between G. intraradices, G. mosseae, and Scutellospora casanea and
found that in addition to a common set of AM-related genes, there were significant
differences in host responses to the different fungal species, although this correlated
with different infection types. Similarly, Gao and colleagues reported that induction
or repression of defense-related genes correlated with the infecting fungi and their
ability to penetrate the root (Gao et al. 2004). AM fungi can form two developmen-
tal patterns, the Arum-type and the Paris-type, the former penetrating with one
hyphae into one arbuscule-containing cell, whereas in the latter, hyphae can grow
from cell to cell and thus penetrate many more cell walls. Increased defense gene
expression was observed mainly in interaction with high fungal penetration rates in
the Paris-type interactions, although analysis of a tomato mutant with reduced
infection suggested that induction of plant defense genes does not necessarily
restrict infection by AM fungi (Gao et al. 2004).
Proteomic analyses of M. truncatula roots in response to the oomycte pathogen
Aphanomyces euteiches have shown a correlation between expression levels of
PR10 (pathogenesis-related) proteins and pathogen infection levels in plant lines
with various levels of resistance (Colditz et al. 2004). This study also showed the
preinfection of roots with mycrorrhizal fungi protects from subsequent pathogen
infection, and that this was accompanied with induction of proteins of the phenyl-
propanoid pathway and proteolytic proteins which could be involved in protection
from pathogens. Subsequent RNAi studies have confirmed that silencing of certain
PR10 genes increased plant resistance to A. euteiches, concomitant with the induc-
tion of a different class of PR proteins in the silenced roots (Colditz et al. 2007).
Gene expression studies of roots responding to infection with endoparasitic
nematodes have demonstrated downregulation of many defense-related genes
(Jammes et al. 2005; Puthoff et al. 2003) including JA biosynthesis genes (Ithal
et al. 2007b). This suggests that nematodes, which move through host roots either
intercellularly (root knot nematodes) or intracellularly though vascular tissue (cyst
nematodes), actively inhibit host defense responses. Thioredoxin peroxidase, a
nematode secreted protein, could mediate reduced defense responses by repressing
formation of reactive oxygen species (Robertson et al. 2000). However, other
studies have reported increased expression of defense- and stress-related genes
(e.g., Alkharouf et al. 2006; Gheysen and Fenoll 2002; Ithal et al. 2007b). Compar-
ative analyses of gene expression changes in susceptible and resistant plants have
identified several candidates for resistance to nematodes, including a glycosyltrans-
ferase in tomato (Schaff et al. 2007), a range of syncytial-specific genes including a
WRKY transcription factor and a receptor-like kinase in soybean (Klink et al.
2007a). In addition, responses of the same soybean species to compatible and
incompatible cyst nematodes have also shown extensive differences in gene expres-
sion in the roots within 12 h, again involving defense-related WRKY transcription
factors (Klink et al. 2007b). A parallel study of gene expression changes in soybean
roots and infecting cyst nematodes has highlighted the extent to which the genomes
of both partners adapt during the interaction, with 429 of 35,611 (1.2%) plant genes
and 1,850 of 7,431 (24%) nematode genes showing altered expression levels during
different stages of infection (Ithal et al. 2007a).
3.3.4 Alteration of Root Development by Microbes
Many rhizoshere microbes can alter the development of roots. Some bacteria
synthesize hormones which can alter root growth, lateral root formation, and cell
division activity. Most of the other microbial signals that alter root development, or
their mechanism of action, remain unknown.
Rhizobia induce new cell divisions inside roots of host plants, which differenti-
ate in an organized fashion to develop into a mature nodule. Purified Nod factors are
sufficient to induce cortical and pericycle cell divisions, and their action has been
linked to the reactivation of key cell-cycle regulators in legumes (Foucher and
Kondorosi 2000). One explanation for their action on cell cycle is their potential to
alter auxin and cytokinin signaling in the root. Both auxin and cytokinin levels and
ratios are crucial for activation of the plant cell cycle (Foucher and Kondorosi
2000). Nod factors alter auxin transport at the site of nodule initiation in indetermi-
nate legumes and this might cause an accumulation of auxin where cell division
occurs (Mathesius et al. 1998b). The alteration of auxin transport is most likely
mediated by an induction of root flavonoids (Mathesius et al. 1998a), and silencing
the flavonoid pathway in M. truncatula by RNAi was shown to abolish the ability of
rhizobia to initiate nodules and to regulate auxin transport (Wasson et al. 2006).
Auxin transport is also altered in the ethylene-insensitive M. truncatula sickle
mutant, and this is linked to hypernodulation (Prayitno et al. 2006b). The involve-
ment of cytokinin in nodulation has been demonstrated in two L. japonicus mutants.
Whereas a mutant defective in cytokinin perception is unable to form nodules
(Murray et al. 2007), a gain-of-function mutant conferring constitutive cytokinin
signaling in the root forms nodules spontaneously (Tirichine et al. 2007).
In M. truncatula, silencing of the cytokinin receptor CRE1 resulted in reduced
nodulation (Gonzalez-Rizzo et al. 2006). If Nod factors alter hormone signaling in
the root, they could be expected to alter other aspects of root development affected
by these hormones, and this has been observed in several studies. The cre1 mutant
has significantly increased numbers of lateral roots (Gonzalez-Rizzo et al. 2006),
and similarly in L. japonicus, overexpression of a cytokinin oxidase (which reduces
cytokinin response) increased lateral root but decreased nodule formation (Lohar
et al. 2004), suggesting a negative role for cytokinin in lateral root and a positive
role in nodule formation. Nod factors and a signal from mycorrhizal fungi also
stimulate lateral root formation and this was shown to require early nodulation
signal transduction genes (Olah et al. 2005). Transcriptome and proteome studies
have identified multiple genes that could be involved in developmental changes
induced by rhizobia, including hormone response genes, transcription factors, and
cell division-related genes, although their function remains unstudied (El Yahyaoui
et al. 2004; Kouchi et al. 2004; Lohar et al. 2006; van Noorden et al. 2007).
Root endoparasitic nematodes cause major developmental changes in host roots
as a result of creating feeding structures (Williamson and Gleason 2003). The
mechanisms of feeding site induction are largely unknown, but results from injec-
tion of nematode secretions into plant cells. Some of the secreted proteins have
been analyzed using a proteomic approach (Jaubert et al. 2002) and at least one
secreted peptide belongs to the plant encoded CLE peptide family that includes
CLAVATA3, a peptide regulating shoot meristem activity in plants (Wang et al.
2005). CLE peptides have recently also been observed in other cyst nematodes and
it has been suggested that they mimic plant ligands for receptors involved in cell
differentiation (Mitchum et al. 2007). Of particular interest in these root–nematode
interactions have been genes involved in the induction of cell division and differ-
entiation in the feeding structures. Microarray, subtractive cDNA cloning, and
SAGE have begun to characterize the extensive changes occurring in host roots
in response to cyst and root knot nematodes (Alkharouf et al. 2006; Bar-Or et al.
2005; Bird 1996; Fuller et al. 2007; Ithal et al. 2007a, b; Jammes et al. 2005; Khan
et al. 2004; Klink et al. 2007a; McCarter et al. 2003; Puthoff et al. 2003, 2007;
Uehara et al. 2007). The induction of cell cycle and auxin and cytokinin response
genes indicates that nematodes activate the plant cell cycle by alteration of hormone
levels (Bird and Koltai 2000; Gheysen and Fenoll 2002; Goverse et al. 2000b).
Interestingly, there are several overlaps in gene expression and hormone changes
between galls and Rhizobium-induced nodules (Favery et al. 2002; Hutangura et al.
1999; Koltai et al. 2001). Concomitant changes in cell wall modifying enzymes and
cytoskeletal proteins are likely also involved in the activation of cell cycle and cell
expansion during giant cell and syncytium formation (Jammes et al. 2005). In
Arabidopsis, a comparative analysis of gene expression in response to root knot
and cyst nematodes revealed similar expression of certain cytoskeletal and organ
development genes which might have a role in formation of both types of feeding
structures, whereas lipid transfer proteins, hypothesized to be involved in cell
expansion and/or organ development, were differentially expressed between the
two interactions (Fuller et al. 2007). Studies on the global responses of hosts to
nematodes (and other microbes) have been limited by the difficulty of collecting
sufficient plant material of infection structures, and the use of laser capture micro-
dissection to collect individual infected cells (Klink et al. 2007a; Ramsay et al.
2004) is a step toward obtaining more localized expression data.
In general, it has been difficult to distinguish responses related to invasion from
those related to development. In future studies, it would be useful to analyze
mutants defective either in invasion or in developmental changes to separate
these effects.
3.3.5 Nutrient Exchange
Endosymbioses with mutualistic bacteria and fungi are formed preferentially under
conditions of nutrient deficiency, in particular of nitrogen and phosphorus, respec-
tively. Both partners of these symbioses play an active part in regulating nutrient
exchange across membranes in the infection structures. Rhizobia invade dividing
cortical cells but remain separated from the plant cytoplasm by the peribacteroid or
symbiosome membrane (derived from the plant plasma membrane). Often several
bacteroids are housed together in a symbiosome, where nitrogen fixation by nitroge-
nase takes place. Leghemoglobin is an abundant protein inside nodules protecting
nitrogenase from oxygen (which inhibits nitrogenase) at the same time as delivering
oxygen to the electron transport chain. Bacteroids are differentiated rhizobia that
show significantly altered gene and protein expression patterns compared with free-
living bacteria (Ampe et al. 2003; Becker et al. 2004; Djordjevic 2004; Djordjevic
et al. 2003; Pessi et al. 2007). Fixed nitrogen is exported to the plant cytoplasm as
amino acids, and carbon, mainly in the form of tricarboxylic acids, are taken up by
the bacteroids (Lodwig et al. 2003; Prell and Poole 2006). Transcript analyses for
functioning root nodules demonstrated a high activity of sucrose breakdown, glycol-
ysis, and carboxylic and amino acid assimilation (Colebatch et al. 2002a, 2004;
Tesfaye et al. 2006). Nodule tissues of plant origin express a large number of nutrient
transporters (carbon, nitrogen sulfate, and potassium), metal-binding proteins, aqua-
porins, ATPases related to nutrient uptake, and osmoregulaton inside the nodule
(Benedito et al. 2006; El Yahyaoui et al. 2004; Kouchi et al. 2004; K€uster et al. 2004;
Manthey et al. 2004). Interestingly, these studies also found a large number of
regulatory proteins that could be important in the ongoing regulation of enzyme
and transport activity inside nodules (Colebatch et al. 2004). Proteomics studies of
the peribacteroid membrane have identified about 100 proteins, including many
transporters, aquaporins, especially of the nodulin 26 family, ATP-ases, signaling
and defense proteins, and endomembrane proteins, which could be a result of the
endocytotic origin of the peribacteroid membrane (Panter et al. 2000; Wienkoop and
Saalbach 2003). Metabolomic approaches confirmed elevated levels of amino acids,
organic acids, and certain sugars in nodules (Barsch et al. 2006; Colebatch et al.
2004; Desbrosses et al. 2005). Because significant amounts of photoassimilates can
be diverted to nodules for nitrogen fixation, it could be expected that plants limit
carbon supply to ineffective (fix-) nodules. Metabolome analysis of fix- nodules
showed that carbon restriction to nodules occurs as a limitation of carboxylic acid
synthesis in nodules, rather than photoassimilate transport to the nodule (Barsch et al.
2006). Sucrose synthase, which acts in unloading and cleavage of sucrose in the
nodule, appears as another important metabolic control point and its repression
led to major transcriptome and metabolome changes in nodules, particularly repres-
sing amino acid synthesis (Baier et al. 2007). Senescing nodules can become a
nutrient source for the plant, and this often coincides with pod filling. Transcriptome
analysis of aging nodules identified many regulatory genes that could be involved
in controlling the senescence process and revealed a role for ethylene, JA, and GA in
nodule senescence (Van de Velde et al. 2006).
Mycorrhizal fungi depend on carbon allocation from their host and create a
carbon sink in the infected roots. This is accompanied by increased expression of
hexose transporters, activation of fungal glycolysis, and subsequent carbohydrate
storage in ectomycorrhizal associations (Nehls et al. 2007). Induction of specific
phosphate transporters is localized to arbuscules and is crucial for provision of
phosphorus to the plant partner and some of these transporters have recently been
cloned (Harrison et al. 2002; Paszkowski et al. 2002). A plethora of other nutrient
and water transporters and enzymes of primary metabolism have been detected in
AM-infected roots using transcript profiling (Hohnjec et al. 2005; Liu et al. 2003,
2007). Combined transcriptome and metabolome approaches have highlighted the
role of metabolites from plastids and mitochondria in AM-infected roots. Amino
acid, fatty acid, and carotenoid metabolism were activated in AM roots both at the
transcript and metabolite level, and phosphate levels were increased (Lohse et al.
2005; Schliemann et al. 2008). A detailed review of genome-wide gene expression
changes relating to nutrient exchange and concomitant cell wall modifications has
recently been published (Balestrini and Lanfranco 2006).
Similar to the symbiotic structures, nematode feeding sites develop into massive
nutrient sinks, although the plant appears to fail to regulate this process. Nematode
feeding site development is accompanied by increases in expression of sucrose
transporters and enzymes of carbohydrate metabolism and water channels and other
transport proteins (Gheysen and Fenoll 2002; Hammes et al. 2005; Jammes et al.
2005; Uehara et al. 2007).
3.3.6 Feedback Mechanisms
The acquisition of nutrients by roots is intimately linked with the available carbon
supply from photosynthesis in the shoot. Therefore, long distance communication is
necessary to balance the extent of symbiosis in the root with carbon supply from the
shoot. Both RL and AM symbioses are limited by a feedback mechanism called
autoregulation (Caetano-Anollès and Bauer 1988). The number of nodules and
arbuscules in the root is regulated by a gene that acts in the shoot and has been
identified as a leucin-rich receptor like kinase (LRR-RLK) from soybean
(GmNARK), L. japonicus (LjHAR1), and M. truncatula (MtSUNN), as reviewed by
Kinkema et al. (2006). Interestingly, this LRR-RLK has high similarity to the Clavata
1 (CLV1) gene from Arabidopsis that regulates shoot meristem activity (Gresshoff
2003). Mutation of NARK leads to supernodulation or super-mycorrhization of the
root and overall plant growth is often stunted. Grafting studies have shown that
autoregulation is a result of a signal initiated in the root upon infection with rhizobia
or mycorrhizal fungi, which is received by NARK in the shoot, and a second signal is
generated that travels back to the root and inhibits further symbiosis (Delves et al.
1986; Gresshoff 2003). So far it is unknown why both symbioses are affected by the
action of NARK, or what the autoregulation signal is. Metabolite analyses of alfalfa
found that flavonoid synthesis is limited in both RL and AM symbioses by the
autoregulation signal, possibly limiting availability of symbioses-enhancing flavo-
noids (Catford et al. 2006). Metabolome analysis also suggested that the accumula-
tion of isoflavonoids inhibitory to fungal germination in AM-infected roots could be
part of the autoregulation system (Cordier et al. 1998; Schliemann et al. 2008). In the
M. truncatula sunn (super numeric nodules) mutant, it was shown that inoculation of
roots with rhizobia causes an inhibition of auxin translocation from the shoot to the
root and that the supernodulation mutant does not show this long-distance auxin
transport inhibition (van Noorden et al. 2006). In addition, sunn had higher levels of
auxin in the inoculation zone of the root, suggesting that auxin is a positive regulator
and long-distance signal in autoregulation (van Noorden et al. 2006). Proteome
analysis of wild type and sunn roots supported this, showing that the large majority
of proteins induced by rhizobia are also auxin-inducible. The study also identified
proteins differentially expressed between wild type and sunn, including PR10 pro-
teins, a protein involved in JA synthesis, a glutathione-dependent peroxidase, and a
trypsin inhibitor (van Noorden et al. 2007). A transcriptome study also found several
defense-related genes differing in expression between sunn and wild type suggesting
a reduced defense response in supernodulating plants (El Yahyaoui et al. 2004).
Proteome analysis of mycorrhizal fungi-infected wild type and sunn roots showed
protein expression changes of two annexins, a narbonin, a quinine reductase, and a
Kunitz proteinase inhibitor (Amiour et al. 2006). Liu et al. (2007) showed differential
expression of several defense related and other genes including an aquaporin in the
uninoculated and inoculated part of roots of a split-root system infected with
mycorrhizal fungi, but also several genes similarly regulated by mycorrhizal fungi
in both split root parts, confirming that mycorrhizal fungi have long-distance effects
on uninfected parts of the plant. Comparison of gene expression changes in leaves of
inoculated soybean wild type and a supernodulation mutant identified over 100
differentially amplified cDNA fragments of which most changed in wild type but
not in the mutant (Lestari et al. 2006). Of particular interest in this study was
differential expression of several receptor kinases and transcription factors that
might be involved in autoregulation. These studies have highlighted the complex

changes occurring in shoot and root in response to rhizobia and mycorrhizal fungi
and how they are affected by the autoregulation signal, yet the signal itself remains
elusive. Proteome analysis of xylem sap of soybean wild type and NARK mutants
identified some proteins that could potentially travel long distances in the xylem,
including a lipid-binding protein and Kunitz proteinase inhibitor, although none of
these differed between wild type and mutant (Djordjevic et al. 2007). In future,
phosphoproteomics might reveal some of the early targets of the receptor-like kinases
that control autoregulation.
3.4 Conclusions and Future Directions
One of the most interesting findings of recent years has been the overlap in the
signaling pathways utilized by rhizobia and mycorrhizal fungi to invade legume
roots, leading to the hypothesis that the more ancient mycorrhizal symbiosis was
the precursor for the more recent interaction of legumes with rhizobia (Kistner and
Parniske 2002; Sprent and James 2007). Furthermore, genomic tools have revealed
evidence that root parasitic nematodes also share signal transduction pathways,
genes and maybe signaling molecules with RL and AM symbioses (Bird and Koltai
2000; Favery et al. 2002; Gheysen and Fenoll 2002; Koltai et al. 2001; Weerasinghe
et al. 2005). Interestingly, genome sequencing projects have revealed aspects of the
evolution of genes involved in root–microbe interactions. Several nematode genes,
in particular cell wall-degrading enzymes, appear to have higher similarity to
bacterial genes than to eukaryotic genes, suggesting horizontal gene transfer
between root-infecting bacteria and nematodes (Scholl et al. 2003). Future chal-
lenges remain to determine which parts of the microbial genomes are necessary for
their symbiotic or pathogenic behavior, and these questions might become clearer
with comparative genomic studies of a growing number of sequenced organisms.
Likewise, it will be interesting to reveal the whole extent to which similar plant
genes are required for infection, signaling, and developmental changes in response
to soil microbes. The current wealth of genes and proteins identified in genomics
studies will need to be tested in functional, e.g., reverse genetic, studies to explain
how they are involved in root–microbe interactions. It would be particularly
interesting to test the effect of specific mutations on the interaction of plants
with a range of microbes to highlight commonalities and differences. For
parasitic interactions, it will be of interest to identify nematode- and infection
structure-specific genes that could be targeted in strategies to increase nematode
resistance in crop plants.
Acknowledgments We gratefully acknowledge funding from the Australian Research Council

(ARC) for funding through the ARC Centre of Excellence for Integrative Legume Research
(CE0348212) and through a Research Fellowship to UM (DP0557692). Due to space limitations,
we regret that we could not include all recent articles in this area.
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Chapter 4
Plant Genetics for Study of the Roles of Root
Exudates and Microbes in the Soil
Aparna Deshpande, Ana Clara Pontaroli, Srinivasa R. Chaluvadi, Fang Lu,

and Jeffrey L. Bennetzen
Contents
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.2 Natural Variation and Mutagenesis in Arabidopsis
to Identify Alterations in Root Exudate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
4.3 Plant Genetic Determination of Natural Variation
in Rhizosphere and Root-Associated Microbes in the Grasses . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4.4 Implications and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
A. Deshpande
Department of Biological Sciences, Purdue University, West Lafayette, IN 47906, USA
and
Department of Biological Sciences, Houghton, MI 49931, USA
A.C. Pontaroli
Department of Genetics, University of Georgia, Athens, GA 30602, USA
and
Estación Experimental Agropecuaria Balcarce, Instituto Nacional de Tecnologı́a Agropecuaria
(INTA) – Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (CONICET), Balcarce CC
276 (7620), Argentina
S.R. Chaluvadi and F. Lu
J.L. Bennetzen (*)
Department of Biological Sciences, Purdue University, West Lafayette, IN 47906, USA
and
and
Department of Biological Sciences, Houghton, MI 49931, USA
e-mail: maize@uga.edu

100 A. Deshpande et al.
4.1 Introduction
Although plants can be grown in sterile soil in aseptic growth chambers, their
natural lives involve an intense and intimate interaction with a vast number of
microbes, especially those found in soils. The number of different bacterial species
in a single gram of soil has been estimated to be anywhere from a few thousand to
many millions, depending on the soil source and the method of analysis (Foster
1988; Schloss and Handelsman 2006; Aislabie et al. 2008), with still-undescribed
species making up a large share of the total. In addition to eubacteria and archae-
bacteria, many species of fungi, protists, and algae are also found in the soil, often in
association with plant roots. The great majority of these soil microbes have not been
studied to any significant degree, partly because conditions for their axenic culture
have not been developed. For instance, only 26 of the approximately 52 identified
major lineages, or phyla, within the domain Bacteria have cultured representatives.
In fact, it is estimated that less than 1% of the bacterial species in the soil could be
grown in culture with current approaches (Leadbetter 2003; Handelsman 2004;
Leveau 2007), and this number is certain to be much lower if one considers that
most rare microbial components of the soil are completely unknown.
Plants actively secrete very large quantities, and a great diversity, of organic
compounds into the soil. Exudation of anywhere from 5 to 60% of total photo-
assimilate has been reported and found to be highly variable across environmental
conditions (e.g., soil type, time of day, soil moisture, temperature) and plant
genotype or growth stage (Bekkara et al. 1998; Groleau-Renaud et al. 1998; Hughes
et al. 1999; Iijima et al. 2000; Aulakh et al. 2001; Garcia et al. 2001; Prosser et al.
2006). The roles of only a few of these compounds are known or guessed at
(Merbach et al. 1999). Citrate is secreted, sometimes in very large quantities, to
help acidify the soil and thereby promote root growth (Jones and Darrah 1994;
Hinsinger et al. 2006), and this compound also helps bind aluminum in the soil,
thereby decreasing its phytotoxic effects (Hoekenga et al. 2003). Some plants have
been shown to exude phenolic compounds that exhibit allelopathic effects like the
sorghum exudate sorgoleone that is an inhibitor of broadleaf and grass weeds at
concentrations as low as 10 mM in hydroponic assays (Nimbal et al. 1996). Many
other compounds, such as amino acids and sugars, are believed to be secreted by
plant roots in order to promote rhizosphere microbial growth (Brimecombe et al.
2001), although the value to the plant of 1% of the rhizosphere microbes are not
known in any system. Specific secreted phenolic compounds have been shown to be
signal molecules that attract root colonization by useful microbes, nitrogen-fixing
bacteria such as Rhizobium, and mycorrhizal fungi (reviewed in Bais et al. 2006).
The question remains, what do most of these soil microbes do? The active
secretion of so much of the fixed carbon produced by a plant suggests that these
microbes are very important to the plants, but this idea is challenged by the
observation that plants can grow efficiently in sterile soil. Of course, plants that
are grown with fertilizers in a controlled environment do not need symbiotic
relationships that yield limiting growth substances, like the fixed nitrogen provided
4 Plant Genetics for Study of the Roles of Root Exudates and Microbes in the Soil 101
by rhizobia or the phosphate access provided by mycorrhizae. Perhaps, a more

frequent value of rhizosphere microbial associations to a plant is exemplified in the
“take-all” disease, where the Gaeumannomyces graminis var. tritici fungus that
infects wheat roots is overcome in the soil by a beneficial bacterial competitor, a
specific isolate of Pseudomonas fluorescens (Thomashow and Weller 1988; Capper
and Higgins 2007). Unlike sterile soil, potential microbial pathogens in field soil
may exist in staggering numbers and variety, and only attraction of beneficial or
neutral microbial competitors of these pathogens to the rhizosphere would provide
comprehensive protection to host plants.
In the absence of the ability to grow most soil microbes in pure culture, it is
difficult to test their possible contributions to plant growth or plant disease. One
cannot simply inoculate the soil with a single microbe and see its effects on a
potential host plant if one cannot first grow that microbe. However, we have
postulated that we can use our control over host plant genetics to accomplish the
same goals of understanding the roles of microbes in the soil (Deshpande 2006). If
one can find mutations in plants, or segregating natural variation, which determines
the presence/absence or abundance of specific rhizosphere microbes, then this
demonstrates a specific relationship between the product of the mutated or varying
plant gene(s) and the biology of the affected microbe. For instance, if one finds a
natural variation for a low level of sorgoleone production, and sees that this causes
the root to no longer be colonized by mycorrhizae, then this indicates that sorgo-
leone is involved in mycorrhizal colonization (Akiyama et al. 2005).
We have been pursuing this approach to use plant host genetics to dissect
plant–microbe interactions in the soil for the last 10 years. This research has
proceeded very slowly because of the need to establish a foundation for the experi-
ments, a very limited tool set, a challenging level of environmental variation in the
experiments, a surprisingly low level of plant genetic variation for rhizosphere
exudates (at least in Arabidopsis thaliana, see below), and the lack of funding for
such research in the absence of compelling preliminary results. However, recent
advances in DNA sequencing technology have offered the possibility that studies of
plant genetic control of microbial interactions in the rhizosphere and root can be
analyzed comprehensively. This chapter describes our initial results with the genetic
and metagenomic analysis of these interactions.
4.2 Natural Variation and Mutagenesis in Arabidopsis

to Identify Alterations in Root Exudate
We used a model dicot angiosperm, Arabidopsis thaliana, as a target for our initial
studies of plant host genetic effects on rhizosphere microbial populations. Because
high pressure liquid chromatography (HPLC) is such a powerful technique to
separate and display low molecular weight organic compounds like phenolics, we
decided to determine reproducible conditions for exudate production by the roots of
Arabidopsis seedlings by scoring the production from seedlings grown under sterile
conditions. Seeds were first surface-sterilized by gently agitating them in a solution

containing two volumes of 0.1% Triton X-100 and one volume bleach. Seedlings
were grown on filter paper set atop moist glass beads for 15 days in Gamborg’s B5
medium at a temperature of 24 C and an artificial light intensity of 100 mE m2 s1.
On day 15, fresh Gamborg’s B5 medium diluted to 5% of its original concentration
was added to the roots, and the medium (now with root exudates) was collected
after 2 days of additional growth. Pools of ~100 seedlings were grown together in
single vials for this analysis because smaller numbers of seedlings did not yield
sufficient quantities of exudates for HPLC analysis. The liquid samples were frozen
and dried in a Beckman lyophilizer and then resuspended in 98% methanol for
reverse phase HPLC analysis. Under these conditions, a broad array of peaks
representing different compounds were observed, and these were not produced by
dead seeds or the growth media in the absence of growing seedlings (Fig. 4.1).
Many of these peaks were found not to be reproducible from experiment to
experiment, however, so a smaller range of peaks was chosen for specific focus.
These six peaks gave qualitatively consistent profiles detected at 360 nm (Fig. 4.2).
These peaks were both consistent across experiments and had the general properties
of phenolics and related compounds that were good candidates as signal molecules.
Having established a reproducible assay system, we then looked at A. thaliana
ecotypes Columbia, Landsberg erecta, Kashmir-1, Wassilewskeja, and Cape Verde
Islands (CVI) for their root exudation of related compounds. Surprisingly, we saw
no dependable variation for the compounds represented by these six peaks on the
HPLC chromatogram. The ecotype CVI was included in this study because, at the
level of DNA markers, it was the most different of any Arabidopsis ecotype
available at that time. Hence, it was not possible to map genes responsible for
variation in these compounds in any of the various mapping populations developed
in Arabidopsis from crosses between these ecotypes.
Having failed to detect useful natural variation for exudate production, we next
investigated the production of the compounds represented by these six peaks in
EMS mutagenized Columbia and Landsberg erecta backgrounds, with M3 seed
provided by Lehle Seeds. Most surprisingly, out of 2,000 M3 populations analyzed,
not a single reproducible variation in any of these peaks was identified. Given the
mutation rate in these EMS populations, we expected that 2,000 M3 would have
provided an average of 1–2 homozygous and 3–4 segregating knockout mutations
per gene for every gene in the Arabidopsis genome. Hence, for the first time in the
history of genetics, we apparently identified a series of biological processes to
produce numerous compounds that are not affected by mutational inactivation of
any single gene. This astounding result remains unexplained.
Because it was expected that many of the compounds in the studied six peaks
were phenolics, we also looked at known mutations in phenolic pathways, including
knockout mutations in fad1-2, fae2-1, gsm1-1, gsm1-2, hy5-1, mur2-1, mur4-1,
mur5-1, and rhd1-1, plus the double mutants fah1-7/tt3-1, tt3-1/ tt7-1, and tt4-1/tt5-
1 (Koornneef 1990; Lemieux et al. 1990; Haughn et al. 1991; Miquel and Browse
1992, Reiter et al. 1997) obtained from the ABRC. In addition, a line exhibiting
transgenic F5H overexpression, generously provided by the laboratory of Dr. Clint
Fig. 4.1 A three-dimensional metabolite profile of root exudates showing the retention time
(X-axis), peak intensity (Y-axis), and the UV range of 200–400 nm (Z-axis). (a) Root exudates
of wild-type Arabidopsis thaliana plants, ecotype Columbia; (b) negative control (growth media
processed as exudate)
Chapple (Purdue University), was also investigated. Arabidopsis lines that were
mutant in these genes were found to not exhibit any qualitative changes in the six
putative phenolic peaks that we focused on throughout this project.
Fig. 4.2 Chromatograms of wild-type Arabidopsis thaliana root exudates showing the six major
peaks detected at 360 nm. (a) Ecotype Columbia; (b) negative control
The rationale of the Arabidopsis studies had been to identify genetically

determined exudate variation and then to follow this up with the characterization
of both the exudate compound(s) affected and the degree to which this variation
altered soil microbial populations associated with the Arabidopsis root. In
the absence of identified genetic variation, such follow-up studies were not
performed.
4.3 Plant Genetic Determination of Natural Variation

in Rhizosphere and Root-Associated Microbes
in the Grasses
After arriving at the University of Georgia (UGA) in 2003, our lab decided to look
at several grass species as targets for the study of root–microbe interactions. These
studies have not yet involved exudates analysis but went directly to a metagenomic
analysis of soil microbes. The soil used was from different UGA fields, but each
experiment involved mixing one field soil source with a uniform potting mixture (to
make roots easier to subsequently extract) and then placing equal amounts of this
mixture in each large pot used in the experimental study. Seeds for host plants were
germinated in these soils, and seedlings were then grown in the greenhouse under
the same conditions for each duplicated or triplicated plant in the experiment. The
assay system has been to sequence either total DNA or 16s ribosomal DNA
amplicons prepared from the soil that clings to an extracted root (“rhizosphere”
or Rh), the microbes firmly attached to a root washed with water (“root-external
microbes” or REM), and the microbes remaining after the root is treated with
chitinase, lysozyme, and various levels of hydrogen peroxide (“root-internal
microbes” or RIM). Of course, the sample termed REM contains both root-internal
and root-external microbes, while the Rh sample is certainly contaminated by
broken root fragments that would yield some root-internal and root-external
microbes.
In order to guarantee that the DNA analyzed would provide a comprehensive
description of the microbes that were present, a vigorous DNA extraction protocol
(http://fgp.bio.psu.edu/methods/ctab.html) was followed. Hence, the DNA extrac-
tion procedure for Rh, REM, and RIM samples yields not just the microbial DNA
but also DNA from any other organisms or tissue fragments that were present in the
sample. Especially in the case of the REM and RIM samples, this meant that there
was a tremendous amount of host plant DNA present. Hence, random shotgun
sequencing of all root-associated samples was mostly an exercise in sequencing the
host plant genome, with yields of 10–20% of cloned DNA (Table 4.1) that was
verified as nonplant. At the time of these analyses, neither the sorghum nor maize
genomes had been fully sequenced, so many of the sequences labeled unknown
could be screened for homology to these genomes once the ongoing sequencing
projects are completed. Regardless, it was clear that this was an expensive route to
pursue for metagenomic discovery.
Because the majority of maize nuclear DNA is methylated at the cytosines in
50 -CG-30 and 50 -CNG-30 sequences, we decided in one experiment to transform all
of our soil DNA into DH5-a because cytosine methylated DNA such as that seen
in maize and other grasses is often destroyed by this Escherichia coli strain (Palmer
et al. 2003). Sequences of the resulting clones provided a significant decrease
in maize DNA, and a significant increase in the percent of bacterial sequences
recovered (Table 4.1) but decreased the amount of mycorrhizal DNA that was
observed (data not shown). Hence, this potential metagenomic enrichment technology
106
Table 4.1 Shotgun sequence analysis of DNA from the plant–soil interface
Host plant Microbial Seq, typeb Number of Percent of sequences with highest homology to DNA from the listed organisms Unknown
species fraction sequences Host plant Moss Eubact. Archaea Fungi Protist Diatom Animal Phage
targeteda sequences
Zea mays Rh+REM+RIM RS 732 88.3 – 3.7 – 2.2 – 0.1 0.4 – 5.3
MF 218 30.2 – 48.6 0.5 6.9 – 0.5 – – 13.3
REM+RIM RS 259 89.2 – 1.9 – 4.6 – – 0.8 – 3.5
MF 249 34.1 – 44.6 – 6.0 – – 0.8 – 14.5
RIM RS 382 89.3 – 1.1 – 1.3 0.3 – – – 8.1
MF 176 43.7 – 46.0 – 2.8 – 0.6 1.7 0.6 4.6
Sorghum bicolor Rh+REM+RIM RS 366 88.3 – 4.6 – 0.5 – – – – 6.6
MF 95 48.4 2.1 36.8 – 4.2 – – – – 8.4
REM+RIM MF 84 76.2 – 13.1 – 3.6 – – – – 7.1
RIM MF 83 44.5 – 30.1 – 1.2 – – 1.2 – 22.9
Sorghum Rh+REM+RIM RS 352 80.7 – 5.1 – 1.2 – – – – 13.1
propinquum MF 89 58.4 – 12.4 – 3.4 – – – – 25.9
REM+RIM MF 83 60.2 1.2 24.1 – – – – – – 14.5
RIM MF 85 51.8 – 21.2 – 1.2 – – – – 25.9
a
Rh þ REM þ RIM rhizospheric þ root-external þ root-internal microbes, REM þ RIM root-external þ root-internal microbes, RIM root-internal microbes
b
RS random shotgun, MF methyl-filtered
A. Deshpande et al.
was abandoned because it was not likely to yield a representative description of the
microbes present in the soil, rhizosphere, or root samples. We also abandoned the
hydrogen peroxide treatment in our RIM purification process because the level of
treatment that we employed (2 min in 3% H2O2) appeared to lead to degradation of
some DNA inside roots (data not shown). Moreover, although hydrogen peroxide
treatment greatly lowered the number of sequences that were recovered from the
extracted DNA, it did not show any obvious effect upon the relative abundances of
classes of eubacteria that were recovered (data not shown). Hence, further investi-
gation of hydrogen peroxide treatment, to identify an appropriate level of exposure
for removing external microbes without damaging root-internal DNA, is warranted
but may not be necessary.
Our first experiments were on the plant species Zea mays (maize), Sorghum
bicolor (sorghum), and S. propinquum (a wild and interfertile relative of sorghum).
The results with random shotgun sequencing of Rh, REM, and RIM microbes
(Table 4.2) indicated that sequences representing many different kingdoms and
phyla of microbes (archaebacteria, eubacteria, fungi, protists), small animals (e.g.,
nematodes and insects), mosses, and even a bacteriophage were present in the data,
although most of the sequences were either from the host plant or of unknown
origin. Interestingly, the organisms in the RIM sample (presumed root-internal
microbes) included protists like Cercozoa (a flagellate protozoan that consumes
bacteria) and the diatom Thalassiosira. These DNA sequences were annotated in
early 2009, when internal funding for this project was exhausted, so reannotation at
this date would be much more informative because additional plant sequences
could be identified, and more of the unknown sequences would be attributed to
many of the additional microbes that have been sequenced since that time.
For reasons of cost effectiveness, we decided to primarily switch to the standard
process for amplification of rRNA genes (Weisburg et al. 1991; Tringe and
Hugenholtz 2008) for microbe identification. This has the disadvantage of a poten-
tial for differential degrees of amplification of different sequences (thus providing a
skewed quantitative description of the microbes present) and the possible lack of
amplification of highly diverged microbes. For cost reasons with the maize and
sorghum samples, only a few eubacterial rRNA sequences were investigated,
providing between 173 and 191 eubacterial reads per duplicated data set. Even
with this limited amount of data, certain patterns were clear. The most abundant
eubacteria both outside and inside roots were from the class betaproteobacteria,
although the deltaproteobacteria were about equally abundant in the REM (root
external) samples for both S. bicolor and S. propinquum (data not shown).
Table 4.2 Analysis of soil and root-associated organisms with 16s, 17s, and 18s rRNA sequences
in switchgrass cultivar “Alamo”
Species Treatment Eubact. Archaea Fungal Protist Animal
phylotypes phylotypes phylotypes phylotypes phylotypes
Switchgrass Rh 668 13 37 19 46
Switchgrass REM 409 3 53 6 5
Switchgrass RIM 284 2 50 8 8
The alphaproteobacteria and gammaproteobacteria were also relatively abundant in

both REM and RIM samples. Such species as the acidobacteria, bacilli, chloroflexi,
clostridia, and deinococci were found both in REM and RIM samples but at
low abundances. The sphingobacteria were of moderate abundance in the REM
samples, but much rarer in the root-internal samples (RIM). Most dramatic, the
Sorghum samples (especially S. propinquum) had a >2X lower percentage of
eubacteria from known classes compared to maize, suggesting that a greater
number/variety of exotic microbes associate with the roots of plants in the genus
Sorghum than with maize.
Recently, we have begun studies of the microbial populations associated with
the candidate biofuel crop called switchgrass (Panicum virgatum). In our first
experiments, we have observed that the Rh, REM, and RIM populations for
switchgrass are quite distinct (Table 4.2). For instance, archaebacteria were very
abundant in the soil sample employed and frequent in the Rh populations but were
very rare in the REM and RIM samples. As seen with maize and sorghum previ-
ously, mycorrhizal DNA was greatly enriched within the roots (the RIM samples).
In general, bacterial, archaebacterial, protist, and animal diversity dropped off
dramatically on and inside the roots compared to the rhizosphere, but detected
fungi were actually more diverse both on and inside roots compared to the rhizo-
sphere (Table 4.2). Preliminary results indicate that different switchgrass cultivars
yield very different abundances for some microbial species (data not shown),
suggesting that host genetics might be used to characterize the factors that deter-
mine the specific host–microbe associations involved.
4.4 Implications and Perspectives
The relationship between plant growth and soil microbes remains one of the great
mysteries in the life sciences. Other than nitrogen fixation by root-internal or root-
associated bacteria (Elbeltagy et al. 2001), only a few cases are known where a soil
microbe provides some benefit to an associated plant (Thomashow and Weller
1988; Bais et al. 2006; Capper and Higgins 2007; Javot et al. 2007; Evelin et al.
2009). However, the tremendous contribution of photosynthate and a great variety
of apparent signaling compounds that are actively released into the soil by roots
indicate that most rhizosphere microbes are intentionally attracted by the plant. The
simplest model for the role(s) of these microbes is protection from disease caused
by that subset of microbes or animals in the soil that can pathogenize or parasitize
plants via their roots. It is striking that the species diversity of microbes in the soil is
orders of magnitude greater than that available to the aerial parts of the plants, yet
soil-vectored/root-targeted pathogens of plants are relatively rare compared to
those that infect above the ground. In one very preliminary experiment, we
observed that greenhouse-grown maize, sorghum, and sunflower were slightly
less vigorous if grown on field-derived soil than they were on sterilized field soil.
Least healthy of all were plants grown on the same field soil that had been treated
with erythromycin, a broad-spectrum antibiotic that should have killed many of the
eubacteria, suggesting that these bacteria provide some nutrients or protection from
other microbes in the soil.
The most surprising results in this study were that no Arabidopsis mutants were
identified for exudate production. There exists the very trivial explanation that the
stocks that we obtained were not actually mutagenized. It is also possible (however
unlikely) that every one of these exudates compounds is synthesized by enzymes
and regulated by proteins that are encoded by redundant genetic pathways. The lack
of natural variation in exudate production by Arabidopsis was also a surprise, and it
reinforces the idea that these compounds are so important that their composition
and approximate levels are fixed within the species. However, a recent study has
found that two Arabidopsis ecotypes in our study (CVI and Landsberg erecta) were
quite different in their exudates profile, and that this strongly affected rhizosphere
microbial composition (Micallef et al. 2009). We have no explanation for the
dramatic difference in conclusions about exudate variability between our results
and those of Micallef and coworkers, other than the differences in the exudate assay
systems employed. It has also been recently observed that some ATP-binding
cassette (ABC) transporter mutants of Arabidopsis lead to altered root secretion
of phytochemicals and significantly altered fungal and bacterial communities in the
rhizosphere (Badri et al. 2009). It is puzzling that such mutations were not detected
in our experiments.
The much-greater diversity of microbes outside the root compared to on the root
(REM) and inside the root (RIM) suggests that there is a much greater diversity of
environments and niches to fill in the soil than within a plant. The absence of
archaebacteria from inside the roots makes sense, given the facts that the great
majority of archaebacteria are extremophiles and that plants (like all other organ-
isms) attempt to maintain a consistently moderate internal environment that is
necessary for the physiology associated with efficient growth and development.
The most promising results to date are the differences observed in microbial
populations associated with different cultivars of switchgrass. The tetraploidy and
near-obligate outcrossing nature of this grass species makes it ideally unsuited for
genetic dissection of any trait, including plant determination of soil microbial
populations. Nonetheless, a perennial plant like switchgrass is particularly depen-
dent on a durable and very efficient root system, so studies in the switchgrass
rhizosphere are important. However, if funding were available, such studies would
probably move much more rapidly if performed in diploid grasses with excellent
genetics, such as maize, rice, or the close switchgrass relative called foxtail millet
(Setaria italica) (Doust et al. 2009).
Acknowledgments We thank Clint Chapple for providing seed, for lab space and facilities to
perform the HPLC analysis, and for his many helpful comments regarding the Arabidopsis
component of this project. This research and preparation of the manuscript were supported by
endowments to the JLB laboratory from Purdue University (Umbarger Professorship) and the
University of Georgia (Giles Professorship and the Georgia Research Alliance), and the switch-
grass studies by the BioEnergy Science Center (BESC), a research consortium funded by the U.S.
Department of Energy.
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Chapter 5
Impact of the Environment on Root Architecture
in Dicotyledoneous Plants
Véronique Gruber, Ons Zahaf, Anouck Diet, Axel de Zélicourt,

Laura de Lorenzo, and Martin Crespi
Contents
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
5.2 Root System Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
5.2.1 Lateral Root Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
5.2.2 Symbiotic Interactions and Legume Root Architecture . . . . . . . . . . . . . . . . . . . . . . . . . 118
5.3 Plasticity: How the Action of the Environment on the Regulation of Gene Expression
Affects Root Growth and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
5.3.1 Spatial Control and Transcriptional Complexity in Response to Stress . . . . . . . . . 123
5.3.2 Establishing Regulatory Networks: TFs and MicroRNAs . . . . . . . . . . . . . . . . . . . . . . 124
5.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
5.1 Introduction
The pattern of lateral root formation is a complex developmental process that is tightly
regulated to achieve efficient nutrient and moisture acquisition from the soil in all land
plants (Osmont et al. 2007). In addition to lateral roots, legume roots are capable to
develop post-embryonically another organ resulting from the symbiotic interaction
with soil rhizobia, the so-called symbiotic nitrogen-fixing nodules. Efficient use
of symbiotic nitrogen fixation in legumes is an important agricultural trait (Stacey
et al. 2006). Common mechanisms affecting lateral roots and Rhizobium–legume
interactions seem to exist to regulate the action of meristems in root tissues to optimize
root growth with a particular soil environment.
V. Gruber, A. Diet, and A. de Zélicourt

Institut des Sciences du Végétal, C.N.R.S., 91198 Gif-sur-Yvette, France
and
Université Paris Diderot Paris 7, Les Grands Moulins, 16 rue Marguerite Duras, 75205 Paris
Cedex 13, France
O. Zahaf, L. de Lorenzo, and M. Crespi (*)
Institut des Sciences du Végétal, C.N.R.S., 91198 Gif-sur-Yvette, France
e-mail: crespi@isv.cnrs-gif.fr

114 V. Gruber et al.
Abiotic stresses impact severely plant development and productivity. To cope

with these environmental stresses, plants have evolved complex cell signaling
pathways in response to environmental stimuli and have acquired plasticity in
metabolic functions and developmental switches to gain a new equilibrium between
growth, development, and survival. In plants, the root system is the primary site of
perception of the soil environment and diverse stresses, including salinity and
drought (Osmont et al. 2007; Nibau et al. 2008).
In this chapter, we describe first the different meristems arising from the root
system in two model plants, Arabidopsis and Medicago, and then discuss common
mechanisms controlling lateral root development and the formation of the symbi-
otic root nodules. In contrast, we will not describe mycorrhizal interactions here,
even though they have an important impact on root nutrition and architecture,
because these interactions do not involve formation of new meristems. We referred
to other nice reviews for this topic (Bending et al. 2006; Osmont et al. 2007;
Reinhardt 2007; Parniske 2008). Additionally, we focus on the role of plant
hormones in lateral root development in Arabidopsis and nodule organ formation
in legumes.
5.2 Root System Development
5.2.1 Lateral Root Development
In most eudicot plants, only primary roots are formed during embryogenesis and
emerge during seed germination. The branching process in roots depends on the
formation of new meristems starting from a limited number of pericycle lateral root
(LR) founder cells (Fukaki et al. 2007). After germination, pericycle cells in the
root, which constitute a cylindrical layer of cells surrounding the central vascular
tissue, become competent to undergo a characteristic program of cell divisions and
expansions to form lateral root primordia (LRP) post-embryonically. The primor-
dium emerges from the primary root by cell expansion particularly apparent in cells
near the base of the primordium. Then, the new LR meristem begins to elongate,
cell numbers increase at the root tip, and the LR emerges from the parental primary
root (Malamy and Benfey 1997; Malamy 2005; Dubrovsky et al. 2006; Osmont
et al. 2007).
In Arabidopsis and most other dicots, LRs are formed only from pericycle cells
overlying the protoxylem poles of the parent root (Barlow et al. 2004). After
stimulation and dedifferentiation of the pericycle founder cells, cell re-entry and
asymmetric cell divisions of pericycle derivatives produce a dome-shaped primor-
dium with a radial organization similar to that of the mature root tip (Dubrovsky
et al. 2000, 2001; Beeckman et al. 2001; Casimiro et al. 2001, reviewed in De Smet
et al. 2006; Osmont et al. 2007). Pericycle founder cells acquire a different
developmental fate during the first stages of lateral root initiation. In dicots, lateral
root founder cells are recruited from the pericycle cells adjacent to the xylem pole
5 Impact of the Environment on Root Architecture in Dicotyledoneous Plants 115
and formed from a minimum of three or six founder cells depending on longitudinal
unicellular or bicellular initiation (Dubrovsky et al. 2001). The subset of cells
associated with the xylem is strongly competent to initiate cell division contrary
to those associated to phloem, which remain quiescent. Indeed, xylem pole pericy-
cle cells, from which founder cells are recruited, carry cytological meristematic
features such as large nuclei, dense cytoplasm, and small vacuoles (Himanen et al.
2004; Parizot et al. 2008).
To gain insight into the specification process, de Smet et al. (2008) performed
live imaging on longitudinal pericycle cell files during lateral root initiation in
Arabidopsis. Time-lapse recordings revealed a repeated cell division pattern com-
posed of two successive rounds of asymmetric cell divisions, generating a central
core of four small cells and two larger flanking cells. To achieve this, the original
pericycle lateral root founder cells undergo an initial asymmetric division to
generate a smaller daughter cell and a larger flanking cell. The latter will undergo
another asymmetric division, resulting in a central core of small cells. Hereafter, the
process of anticlinal asymmetric cell divisions stops, and the two central cells
change their axis of division by 90 and divide periclinally. The flanking and the
adjacent undivided pericycle cells undergo few or no anticlinal divisions and will
only contribute modestly to the flanks of the primordium (Fukaki et al. 2007).
Potential factors involved in regulating asymmetric cell division pattern were
identified using transcript profiling on sorted pericycle cells undergoing lateral root
initiation (de Smet et al. 2008). Among them, the receptor-like kinase Arabidopsis
CRINKLY4 (ACR4) appears as a key factor both in promoting formative cell
divisions in the pericycle and in constraining the number of these divisions once
organogenesis has been started. ACR4 is transcribed specifically in the small
daughter cells after the first asymmetric pericycle cell division. ACR4 represses
supernumerary formative divisions of root cells, both in pericycle cells during
lateral root initiation and in the columella in the root apex. ACR4 signaling is
therefore a critical homeostatic mechanism in mediating formative divisions in
pluripotent root tissue during organogenesis and might act both cell- and non-cell
autonomously. Cell autonomously, ACR4 might be required for correct specifica-
tion of lateral root primordia cells whereas non-cell autonomously, ACR4 signaling
might prevent neighboring pericycle cells from becoming triggered for LR initia-
tion. Although specification of founder cells is a key event in postembryonic organ
formation, the mechanisms underlying this process are largely elusive. The restric-
tion of formative cell division to a few pericycle cells and the specification of stem
cell identity in the branching process in roots are not yet well understood.
Auxin promotes organ formation (Reinhardt et al. 2000, 2003; Tanaka et al.
2006), and locally increased levels of auxin response have been reported to mark
positions of organ initiation and distal tips of developing organ primordia (Benkova
et al. 2003; Heisler et al. 2005; Laskowski et al. 2006). LR initiation has since long
been considered to occur after re-entry of pericycle cells into the cell cycle from an
arrested G2 phase through the action of auxin (Blakely and Evans 1979; Laskowski
et al. 1995; Malamy and Benfey 1997). However, experimental evidence argues
against this dedifferentiation concept. Studies in young apical region of the
Arabidopsis root, just above the elongation zone, emphasize the mitotic compe-
tency of the pericycle and counter the G2 re-entry hypothesis as most of the
pericycle remains in the G1 phase, with only the xylem pole pericycle cells
progressing from G1 to G2 phase (Beeckman et al. 2001). Correspondingly,
xylem pole pericycle cells continue to cycle without interruption after leaving the
root apical meristem (Dubrovsky et al. 2000). Taken together, these data question
the differentiated nature of pericycle cells and argue for the concept of a mono-
layered extended meristem. Nevertheless, new LRs can also initiate in more mature
parts of the root, between earlier ones, which necessitate therefore a dedifferentia-
tion and cell cycle re-entry for pericycle cells (Casimiro et al. 2003).
In Arabidopsis, the pericycle has been shown to have competence to divide due
to the constitutive expression of at least two core cell cycle genes, the cyclin-
dependent kinase CDKA;1 and the cyclin CYCA2;1 (Beeckman et al. 2001; Roudier
et al. 2003). Furthermore, pericycle cells continue to divide at the xylem pole, but
most of the divisions do not result in LR initiation and are purely proliferative.
Accordingly, based on the genetic and phenotypic characterization of the Arabi-
dopsis alf4-1 mutant (Celenza et al. 1995), which is not capable to initiate any LR.
DiDonato et al. (2004) have shown that ALF4 is required to maintain the pericycle
in a mitosis-competent state needed for LR formation. The competent state appears
to be a prerequisite for the very first asymmetric divisions, because no such
divisions and no mitotic cyclin CYCB1;1 expression are observed in the mutant.
Moreover, Himanen et al. (2002) reported that the KRP2 gene, encoding the CDK
inhibitor of the G1- to S-phase transition, is strongly expressed in non-dividing
protoxylem pericycle cells. Overexpression of KRP2 decreases the number of LRs
regulating negatively the cell cycle progression during pericycle reactivation. The
G1- to S- phase is therefore one of the targets for auxin-mediated LR initiation
(Himanen et al. 2002, 2004 , Vanneste et al. 2005).
Despite the importance of the cell cycle in LR initiation, increasing the mitotic
index in roots or forcing excessive cell divisions in the pericycle does not stimulate
LR initiation or morphogenesis (Vanneste et al. 2005; Wang et al. 2006). Lateral
root initiation takes place only when cell cycle activation is accompanied by cell
fate re-specification of pericycle cells triggered by auxin-induced degradation of the
SLR/IAA14 protein. Lateral root founder cell specification and patterned cell
division in the pericycle can be separated both temporally and genetically indicat-
ing that the primary event during LR primordium initiation may not be exclusively
an auxin-induced activation of the cell cycle (De Smet et al. 2006). Dubrovsky et al.
(2008) have shown that an increase in auxin levels and signaling in individual
pericycle cells always accompanies lateral root organogenesis, and that such
increases are sufficient for the acquisition of lateral root founder cell identity.
This process is not directly coupled to subsequent division of the founder cells, as
the specification event can be genetically separated from the patterned division
during primordium morphogenesis. The local accumulation of auxin in individual
xylem pericycle cells could result from either directed transport or local synthesis
and serves as a local instructive signal for cell fate reprogramming. This mechanism
of local auxin maxima can thus select given pericycle cells and convert them into
founder cells, thereby determining a spatial pattern of lateral root formation. A

model whereby auxin serves as a morphogenetic trigger for LR initiation was
proposed (Dubrovsky et al. 2008). Interestingly, using the DR5::GUS auxin
reporter line, De Smet et al. (2007) have reported that an oscillating auxin response
maximum in the basal region of the meristem seems responsible for priming
pericycle cells for lateral root initiation suggesting that early events in the life of
a pericycle cell might affect its future competence for lateral root formation.
The polar auxin transport is required to form lateral roots as demonstrated with
mutants defective in polar auxin transport failing to produce lateral roots (Benkova
et al. 2003; Geldner et al. 2004). Roots use a transcellular auxin signaling network
designed to synchronize lateral root development and emergence processes. The
AUX/IAA-dependent repositioning of auxin efflux carriers toward the tip of the
newly formed LR is linked to an important change in the direction of auxin flow
favoring a LR growth perpendicular to the primary root (Benkova et al. 2003; Sauer
et al. 2006). The PIN and AUX/LAX (such as AUX1) classes of auxin transport
proteins have key roles in transmitting or localizing the inductive IAA signal,
respectively (Kramer 2004). Although PIN class of auxin efflux carrier expressed
by the lateral root facilitates the transmission of the inductive IAA signal, the ability
to localize the auxin response to cells directly overlaying lateral root primordia is
dependent on the auxin influx carrier LAX3 (Swarup et al. 2008). Auxin induces the
expression of LAX3 in cortical and epidermal cells directly overlaying new LR
primordia creating a positive-feedback loop. LAX3-expressing cells will become
more efficient sinks for auxin. LAX3 therefore functions to amplify the signal
emitted by the lateral root primordium tip while limiting its action to a few cells
in close proximity with this auxin source.
Hirota et al. (2007) reported that PUCHI acts downstream of auxin signaling and
contributes to lateral root morphogenesis through affecting the pattern of cell
divisions during the early stages of primordium development. Indeed, the expres-
sion of PUCHI is regulated by auxin through ARF transcription factors (TFs)
during the early stages of LRP development, in particular ARF7 and ARF19,
which are key regulators of LR initiation, whose activities are negatively regulated
by the IAA protein SLR/IAA14 (Fukaki et al. 2005; Okushima et al. 2005).
Moreover, ectopic expression of a stabilized mutant IAA14 protein in early LRPs
results in the formation of disorganized primordia, suggesting that the normal auxin
response mediated by Aux/IAA signaling is required for proper patterning of LRP
(Fukaki et al. 2005). Microarray analyses have indicated that the induction of
PUCHI expression by auxin does not occur in the slr-1 or arf7 arf19 mutant
background (Okushima et al. 2005; Vanneste et al. 2005). Although it is not
known whether the ARF7 and ARF19 proteins are involved not only in LR
initiation but also in subsequent morphogenesis of the LRP, it is possible that
PUCHI expression may be directly regulated by these ARF proteins. Alternatively,
expression of PUCHI may be regulated by other unknown ARF proteins that are
activated by auxin during early LRP development.
In addition to auxin, other hormone signals are important for LR emergence as
recently reviewed by Fukaki and Tasaka (2009). Lateral root growth is regulated
antagonistically by auxin and cytokinin. Cytokinin is a negative regulator of LR

formation in many plant species, including Arabidopsis and Medicago (Werner
et al. 2003; Gonzalez-Rizzo et al. 2006; Li et al. 2006; Laplaze et al. 2007).
Cytokinin signaling is repressed in xylem-pole pericycle cells (Mahonen et al.
2006). Transactivation of the Arabidopsis cytokinin-degrading enzyme cytokinin
oxidase 1 in lateral root founder cells results in increased lateral root formation.
Laplaze et al. (2007) observed that cytokinins perturb the expression of PIN genes
in lateral root founder cells and prevent the formation of an auxin gradient that is
required to pattern lateral root primordia.
Ethylene has a stimulatory effect on adventitious root formation in many plant
species (Clark et al. 1999). Negi et al. (2008) reported that ethylene negatively
regulates Arabidopsis LR formation by altering auxin transport. This ethylene-
enhanced IAA transport depends on AUX1, an IAA influx carrier, because the
aux1-7 mutant is insensitive to ethylene as an enhancer of acropetal and basipetal
IAA transport, and thus for the inhibition of LR formation.
Abscisic acid (ABA) can reversibly block meristem activation post-emergence
by inhibiting the cell cycle gene expression necessary for meristem activity, leading
to LR growth arrest (De Smet et al. 2006). Interestingly, ABA appears to have the
opposite effect on LR emergence in legumes, stimulating LR formation in Medi-
cago (Liang and Harris 2005). The Medicago latd mutant has a reduced root surface
area with short primary roots, arrested LRPs, and disorganized meristems (Bright
et al. 2005). However, exogenous application of ABA rescued at least partly the latd
phenotype, and latd mutants seem to be impaired in ABA perception or signaling
(Liang et al. 2007).
Lateral root formation is modified to optimize the growth of the root system in a
particular soil environment. LRP initiation and emergence are separable processes
providing therefore greater plasticity to the root system (Dubrovsky et al. 2006).
Cells in the parent root overlaying new lateral root primordia actively participate in
organ emergence. In several plant species, cells from root tissues overlaying new
primordia are recruited to form a temporary root cap that assists organ emergence
(Casimiro et al. 2003; Dubrovsky and Rost 2003; Ivanchenko et al. 2006). How-
ever, the principles that govern the longitudinal positioning and spacing of lateral
root primordia are not yet understood (Malamy 2005).
5.2.2 Symbiotic Interactions and Legume Root Architecture
Legume roots are capable to interact symbiotically with nitrogen-fixing soil bacteria
known as rhizobia, to form the so-called root nitrogen-fixing nodules. In this symbi-
osis, compatible rhizobia and plant partners recognize each other through the
exchange of chemical signals (Limpens and Bisseling 2003). Host plants produce
compounds acting as inducers of the bacterial nod genes, whose products are
involved in the synthesis and secretion of a specific rhizobial lipochitooligosaccharide
signal named the Nod factor. The Nod factor signal triggers a series of host
responses, culminating in the development of the root nodule, in which rhizobia

convert atmospheric nitrogen to nitrogen-containing compounds (Oldroyd and
Downie 2008). The signal perception by the host initiates epidermal infection
and stimulates the cortical cell divisions that give rise to the first cells of the new
root-derived organ. In Medicago truncatula and other temperate legumes, inner
cortical cells dedifferentiate and proliferate, whereas in Lotus japonicus and other
tropical legumes outer cortical cells are recruited (Stacey et al. 2006). Other bacte-
rial surface components, such as exopolysaccharides or lipopolysaccharides, are
also required for the elongation of infection threads and further stages of nodulation
(Jones et al. 2007).
Nodule initiation involves two primary processes: root infection and nodule
primordium induction. These processes occur predominantly in the developmen-
tally receptive zone-of-elongation in legume roots. Rhizobia gain entry into the root
tissues via plant-derived infection threads which route the bacteria toward the
developing primordium (Limpens and Bisseling 2003; Fournier et al. 2008). In
tropical-type nodules, the meristematic activity of the nodule occurs only at early
stages of organogenesis, leading to round-shaped nodules with determinate growth.
The meristem is transient, and all the primordia cells differentiate into mature
nitrogen-fixing nodule cells. In temperate legumes exhibiting indeterminate
nodules, Rhizobium-derived Nod factors stimulate pericycle, endodermal, and
inner cortical cells at proximal xylem poles to enter the cell cycle, divide, generate
new symplasmic connections with phloem cells in the stele, and form a primordium
containing pluripotent stem cells (Timmers et al. 1999; Complainville et al. 2003).
In this section, we will only discuss common mechanisms affecting LRs and
Rhizobium–legume interactions. Indeed, nodules and roots share many aspects of
their development consistent with the theory that nodulation may have evolved
from pre-existing mechanisms dealing with lateral root formation (Hirsch and
LaRue 1997; Mathesius et al. 2000; Mathesius 2003; Ferguson et al. 2005).
Symbiotic nodules and LRs form adjacent to xylem poles, develop meristems, and
emerge through various cell layers from the primary root. However, unlike LRs,
legume nodules lack a root cap and have a peripheral stem-like vasculature, rather
than the central vasculature of roots. In addition, nodule and LR primordia are formed
primarily from different tissues: the nodule from cortex and LRs from pericycle
(Oldroyd and Downie 2008). Nevertheless, pericycle cells are activated and divide
during nodule formation in M. truncatula (Timmers et al. 1999; Complainville et al.
2003), suggesting that the pericycle is at the origin of both organs. A further similarity
between nodule and lateral root development in legumes is the involvement of
cortical cells in the formation of lateral roots (Oldroyd and Downie 2008). Cortical
cells activated during lateral root formation can be induced to form nodule primordia
in mature regions of the root in white clover (Mathesius et al. 2000). Thus, the same
root tissue layers are involved in nodule and lateral root development in legumes, but
to different degrees. Differences in the ontogeny of lateral roots and indeterminate
nodules may be more quantitative than qualitative, with divisions of the inner
cortex providing the bulk of the nodule primordium, whereas predominantly pericy-
cle-derived cells compose the LR primordium. Indeed, roots treated with auxin
transport inhibitors lead to the formation of nodule-like structures with some histo-
logical traits typical of lateral roots in alfalfa and pea (Hirsch et al. 1989; Scheres
et al. 1992).
Insight in the cellular origin of nodule and LR has been obtained through genetic
approaches. In the homeotic mutant cochleata of Pisum sativum, hybrid structures
between nodules and roots are formed. The organs start as nodule, but once the
meristem is formed (characteristic of indeterminate nodules), this meristem turns
into a lateral root. These cochleata nodules appeared functional (able to fix nitro-
gen) and contained a root cap, a LR-like meristem, with the peripheral vasculature
leading to a central vasculature and root hairs, similar to a LR (Ferguson and Reid
2005). These pea mutants are also deficient in gibberellins (GAs) and the reduction
in lateral root and nodule formation could be complemented by exogenous applica-
tion of GAs, suggesting therefore a role for GAs in both type of legume root-derived
organogeneses (Ferguson et al. 2005). The existence of intermediate lateral organs
known as root nodule hybrids in certain legumes or following inoculation with
specific Rhizobium strains further supports the fact that nodule formation evolved
from developmental pathways activated during lateral root formation (Ferraioli
et al. 2004). However, these root nodule hybrids differed morphologically from
those typically detected in the cochleata mutant, as the nodule zonation pattern and
multiple root, nodule and callus structures characteristic of cochleata hybrids were
not observed. In bean, ectopic roots from abortive nodule primordia develop after
infection with different Rhizobium etli mutants called “root inducer” (RIND)
affected in different anabolic pathways (Ferraioli et al. 2004). These mutants
induced a wild-type early sequence of morphogenetics events, including root hair
deformation and nodule primordia development. Later on, however, from the
resulting root outgrowths, instead of nodules, one or more ectopic roots (spaced
closely related and agravitropic) emerged.
The identification of common genes involved in both types of root-derived
organogenesis revealed common regulatory pathways. One example reported by
Wopereis et al. (2000) is the HAR1 (for hypernodulation aberrant root formation)
gene of Lotus japonicus which is involved in the regulation of lateral root and
nodulation numbers and is a shoot-derived trait. This gene codes for a Clavata
receptor-like kinase involved in the regulation of meristem number and nitrate
regulation (Oldroyd and Downie 2008). In addition, analysis of the latd (for lateral
root organ defective) mutant revealed that the LATD gene is required for both lateral
root and nodule development, as well as for maintenance of the primary root
meristem. The latd mutant plants initiate LRs and nodule formation but do not
complete their development resulting in immature, non-nitrogen-fixing nodules and
short bump-like LRs. LATD provides therefore a strong evidence for shared genetic
components between nodule and LRs (Bright et al. 2005). Exogenous ABA rescues
not only meristem organization of latd primary and lateral roots but also meristem
function, restoring cell division and local inhibition of differentiation (Liang et al.
2007). This suggests a direct role for ABA in meristem function and organization in
legume roots as well as in a later step of nodule formation. Secondary root organo-
genesis has also been shown to be controlled by a cytokinin receptor homolog,
MtCRE1 (Gonzalez-Rizzo et al. 2006). Down-regulation of MtCRE1 leads to cyto-

kinin-insensitive roots, which show an increased number of LRs and a strong
reduction in nodulation, supporting interactions between lateral root and nodulation
pathways. This suggests a cross-talk between cytokinin signaling pathways and
development of root lateral organs in legumes (Frugier et al. 2008). Further evidences
for cross-talk between symbiotic nodule and LR developmental pathways have been
obtained through the identification of genes, such as the AUX1-like genes MtLAX1,
MtLAX2 (de Billy et al. 2001), MtANN1 (De Carvalho-Niebel et al. 2002), Medicago
sativa cyclinA2;2 (Roudier et al. 2003), and the early nodulin (enod) genes ENOD11,
ENOD12, and ENOD40, that are highly expressed in both developing nodules and
LRs (Stacey et al. 2006). For example, during LR and nodule development, the
MtLAX genes are expressed in the primordia, particularly in cells that are probably
derived from the pericycle. At slightly later stages, these genes are expressed in the
regions of the developing organs where the vasculature arises consistent with the
involvement of MtLAX genes in local auxin transport. Auxin seems required at two
common stages of lateral root and nodule development: formation of the primordia
and differentiation of the vasculature (de Billy et al 2001; Mathesius 2008). Finally,
the discovery of microRNAs (miRNAs) as post-transcriptional regulators of many
developmental processes, including the formation of vascular tissues (Voinnet 2009),
suggested a possible involvement in legume root architecture. Recently, overexpres-
sion of MtMIR166 in M. truncatula was shown to perturb the organization of root
vascular bundles and increased the number of xylem and phloem poles in roots. This
consequently reduced the number of symbiotic nodules and lateral roots generated
from these roots (Boualem et al. 2008) and was linked to MtMIR166-mediated post-
transcriptional regulation of several HD-ZIP III genes in roots and nodules.
Both symbiotic interactions and soil environmental stresses can profoundly
affect the growth and development of the root and influence the final architecture
of the root system.
5.3 Plasticity: How the Action of the Environment

on the Regulation of Gene Expression Affects
Root Growth and Development
Plants are exposed to a plethora of stress conditions throughout their life cycle. The
two major environmental constraints that currently reduce plant productivity are
drought and salinity. More than 10% of arable land is affected with desertification
and salinization rapidly increasing on a global scale the decline of average yields
for most major crop plants (Bray et al. 2000; Botella et al. 2005). Exposure to both
stresses triggers many common reactions in plants including cellular dehydration
and removal of water from the cytoplasm into the extracellular space resulting in a
reduction of the cytosolic and vacuolar volumes (Verslues et al. 2006). Plants have
evolved complex cell signaling pathways activating metabolic functions and devel-
opmental switches to permit their adaptation to these conditions (Shao et al. 2006;
Umezawa et al. 2006; Yamaguchi-Shinozaki and Shinozaki 2006; Sreenivasulu
et al. 2007). The regulatory circuits include stress sensors, signaling pathways
comprising a network of protein–protein interactions, TFs and promoters, and
finally the output proteins or metabolites. A critical step controlling stress responses
involves thus transcriptional regulation, generally mediated by TFs that may govern
and coordinate the expression of large groups of genes. Plant genomes dedicate a
large number of their coding sequences to TFs reaching about 5.9% (>1,500 TF
genes) in the fully sequenced Arabidopsis genome (Riechmann et al. 2000). In
legumes, extensive sequencing highlighted around 2,000 TFs per genome, less than
1% of them genetically characterized (Udvardi et al. 2007).
Roots are in direct contact with the soil and hence are primary sites for percep-
tion of the soil environment. Abiotic stresses have the ability to elicit morphologi-
cal, structural, and physiological responses to an unfavorable environment in root
growth in order to maximize the acquisition of resources, a property linked to the
so-called root developmental plasticity (Lynch and Ho 2005). TF networks are
known to control root cell identity during development and adaptation to abiotic
stresses also in roots (Montiel et al. 2004; Nibau et al. 2008). The development of
genomic resources and information for model species as Arabidopsis thaliana,
Medicago truncatula, and Lotus japonicus increased considerably the analysis of
TF gene expression on a global genome-wide scale based on their regulation in
response to abiotic stresses (Chen and Zhu 2004; Maggio et al. 2006; Tuteja 2007),
including available publicly databases (e.g., Genevestigator, Ma et al. 2006).
In fact, microarray studies revealed large-scale changes in the transcriptome in
response to specific abiotic stresses (Kreps et al. 2002; Seki et al. 2002; Jiang and
Deyholos 2006; Dinneny et al. 2008). In the model legume M. truncatula, micro-
arrays covering 16,000 genes revealed more than hundred TF genes responding to
early salt stress in root apexes (Gruber et al. 2009). In chickpea, the application of
SuperSAGE (Serial Analysis of Gene Expression) technology to profile transcripts
of drought- and salt-stressed roots from chickpea identified TF genes exclusively
expressed under both stresses, but not in non-stressed controls (Molina et al. 2008).
Several reports have shown a role of TFs as major modulators of stress responses
as salt and drought, in roots. Although the WRKY-type TFs are involved in multiple
abiotic stress responses, the expression of GmWRKY13 in transgenic plants showed
a higher sensitivity to salt and mannitol stress as well as an increase in LRs when
compared to wild-type plants (Zhou et al. 2008). In contrast, GmWRKY54 confers
salt and drought tolerances possibly through the regulation of TFs like DREB 2A
(drought-responsive element binding factor 2A) and STZ/Zat10 (Yamaguchi-
Shinozaki and Shinozaki 2005). Hence, GmWRKY genes play differential roles in
abiotic stress tolerance, and GmWRKY13 may function in both lateral root develop-
ment and abiotic stress responses. In addition, a subclass of APETALA2 (AP2) –
and ethylene-responsive element-binding protein – type TFs, such as DREB2A,
expressed in all root cell layers under salt stress conditions controls semi-ubiquitous
responses to abiotic stresses (Sakuma et al. 2006). Potential direct targets
of DREB2A up-regulated by salt have been identified. Another AP2/ERF-like
(APETALA 2/Ethylene Responsive Factor-like) transcription factor identified via
a gain-of-function Arabidopsis mutant hrd-D is the HRD gene. Overexpression of
this gene improves water use efficiency, drought resistance, and salt tolerance.
This mutant has roots showing enhanced strength, increased branching patterns,
and more cortical cells, accompanied by increased expression of abiotic stress-
associated genes (Karaba et al. 2007). Tolerance to salinity and osmotic stress is
also observed in transgenic tobacco expressing CAP2 (C. arietinum AP2), possibly
because of a large increase of the root system and LRs (Shukla et al. 2006). In
legumes, overexpression of MtZPT2-1 TF genes, linked to recovery processes in
transgenic Medicago roots, allows growth under restrictive salt stress conditions
(Merchan et al. 2007; de Lorenzo et al. 2007). This gene may activate specific
genetic programs linked to the adaptation of legume roots to salt stress. A vascular-
specific bZIP (basic region/leucine ZIPper motif), representing a novel root-specific
transcription factor, is also involved in coordinating gene expression in response to
water-deficit stress in Phaseolus species (Rodriguez-Uribe and O’Connell 2006).
Endogenous phytohormones and regulatory genes sensing the soil environment
may interact to adapt root architecture (Jovanovic et al. 2007). For example,
repressing auxin-induced responses together with enhancement of cytokinin sensi-
tivity may have profound effects on recovery responses after salt stress by limiting
primary root growth, controlling the emergence of lateral roots or the root apical
dominance (Malamy 2005; Aloni et al. 2006; Merchan et al. 2007; Ditengou et al.
2008; Huang et al. 2008; Wolters and J€ urgens 2009). A cross-talk between phyto-
hormone signaling and stress responses in roots was observed for the AtNAC2 TF
(He et al. 2005). It is up-regulated by salt stress and its overexpression in transgenic
Arabidopsis plants results in increased LR formation. This gene is also up-regulated
by ethylene, auxin, and ABA, and its induction by salt is compromised in auxin and
ethylene signaling mutants. On the other hand, the enhanced drought tolerance
conferred by MYB15 overexpression in Arabidopsis seems to be associated to
increased ABA biosynthesis and signaling, which results in greater expression of
several stress-responsive genes and lower water consumption (Ding et al. 2009). In
addition, overexpression of DREB1/CBF also increases the tolerance of transgenic
plants to freezing, drought, and salt stresses (Shinozaki and Yamaguchi-Shinozaki
2000; Sakuma et al. 2002; Fujita et al. 2005) and regulates ABA-independent gene
expression in response to drought and cold stress. Abscisic acid and drought stress
have similar and probably synergistic effects on LR development. Several drought
inhibition of lateral root growth (dig) mutants have enhanced responses to ABA
and are also drought tolerant, whilst others have a reduced LR-inhibition response
to ABA and are drought sensitive (Xiong et al. 2006).
5.3.1 Spatial Control and Transcriptional Complexity

in Response to Stress
Knowledge about responses to abiotic stresses of model plants, such as Arabidopsis

and Medicago, has accumulated during the past decade, based on large-scale
mutant analyses and genome-wide transcript profiles at organ or tissue levels.
These approaches give unrefined localization of gene expression and a few data are
available to correlate stress-related transcript changes and cell-specific gene expres-
sion in an organ.
Ma and Bohnert (2007) analyzed tissue-specific response to stress by integrating
diverse large-scale datasets in which cell type-specific and growth stage-specific
gene expression in Arabidopsis roots was recorded. They combined three types of
data analyzing genome-wide expression profiles modulated by a number of stress
conditions, regulatory cis-elements in promoters, and cell-specific and develop-
mental age-specific root transcripts and their reaction to stress. Among the probes
printed on the Affymetrix chip, 12,360 are considered to be present in at least one of
the three developmental stages of the root: the root expansion growth region (stage 1),
the region of maximum elongation (stage 2), and the root maturation region
(stage 3) also dissected in different cell lineages (lateral root cap, epidermis, cortex,
endodermis, and stele). Among these genes expressed in roots, 5,963 exhibit
statistically significant changes in gene expression during stress. Root-specific
genes down-regulated by abiotic stress are highly expressed in stage 1 root cap
and epidermis under optimal conditions, whereas other genes up-regulated by stress
are expressed in the stage 3 stele, endodermis, and cortex. Thus, complex regulatory
mechanisms can be dissected through intersections of stress-responsive and cell-
specific profiles to identify how cell files are affected by abiotic stresses. Recently,
Dinneny et al. (2008) characterized the transcriptional response to high salinity of
different cell layers and developmental stages of the Arabidopsis root, showing a
highly constraint of transcriptional responses by developmental parameters. Several
tissues tend to be highly responsive as 48% of salt-responsive genes are regulated in
the cortex, 28% in the stele, and 31% in the epidermis. The transcriptional changes
lead to the differential regulation of specific biological functions in subsets of cell
layers which, for certain cases, could be correlated with observable physiological
changes. Known stress pathways primarily control semi-ubiquitous responses, and
mutants disrupting epidermal patterning were used to reveal cell layer-specific and
inter-cellular effects.
A major finding arising from these reports is that cell identity determines the gene
pool that is regulated during stress, as reflected by the high degree of cell specificity
in functional gene categories. This specificity requires maintenance of cell fate
during stress, which is probably ensured by a transcript cohort enriched in cell-
identity genes that remains unaffected by environmental stress (Laurentius et al.
2008). Environmental stimuli combined with cell- and developmental-stage-specific
profiling enable the identification of high-confidence transcriptional modules.
5.3.2 Establishing Regulatory Networks: TFs and MicroRNAs
Even though TFs are central in the regulation of development and stress responses,
post-transcriptional events regulated by miRNAs, e.g., mRNA degradation or
translational inhibition, have also emerged as playing crucial roles in regulating
gene expression (Sunkar et al. 2007; Voinnet 2009). The interaction of miRNAs
and TFs may determine regulatory networks controlling the transcriptome, and
examples have been found to affect root developmental and stress responses.
Lateral root emergence is promoted by auxin signals transmitted by the NAC1
TF (Xie et al. 2000). To study the regulation of the target NAC1 mRNA by miR164,
Guo et al. (2005) manipulated miR164 levels or expressed a miRNA cleavage-
resistant version of NAC1 mRNA in plants. Apparently, miR164 functions as a
negative regulator of auxin-mediated lateral root development by controlling NAC1
mRNA levels and is induced by auxin. This suggests that miR164 mediates the
rapid degradation of NAC1 mRNA to attenuate and terminate auxin signaling. In
addition, disrupting miR160 regulation of ARF17 (Auxine response factor 17)
increases the target ARF17 mRNA levels and leads to severe developmental
abnormalities, including root defects (Mallory et al. 2005). This indicates a critical
role of miR160-directed regulation of ARF17 which seems a transcriptional regu-
lator of GH3-like early auxin-response genes. The Arabidopsis auxin response
factors ARF10 and ARF16 are also targeted by the miR160 and control root cap
cell formation promoting columella cell production (Wang et al. 2005). Indeed,
MIR160 overexpressing plants, in which the expression of ARF10 and ARF16 is
repressed, and the arf10-2 arf16-2 double mutants display the same root tip defect.
They show uncontrolled cell division and blocked cell differentiation in the root
distal region, a tumor-like root apex and loss of gravity sensing. Moreover, auxin
and miR160 regulate the expression of ARF10 and ARF16 genes independently,
generating a pattern consistent with root cap development. Recently, Gifford et al.
(2008) report cell-specific data revealing responses that suggest a coordinated cell-
specific regulation of a transcriptional circuit mediating LR outgrowth in response
to nitrogen via microRNA167 targeting ARF8, one of the pericycle-induced ARFs.
The miR167a, b is expressed specifically in the pericycle and LR cap along with
ARF8, but, consistent with an antagonist effect on ARF8, is repressed in both tissues
in response to nitrogen. Thus, ARF8 offers a link between environmental nutritional
inputs and auxin-mediated plasticity of lateral root architecture. In Medicago, we
mentioned that MIR166 targets a subset of class-III homeodomain–leucine zipper
(HD-ZIP III) TF to regulate LR and root nodule formation (Boualem et al. 2008) as
well as affect vascular bundle patterning. Furthermore, Combier et al. (2006)
showed that miR169-mediated regulation of Medicago MtHAP2-1 expression
leads to a critical spatial and temporal restriction of this TF to the nodule meriste-
matic zone, thereby allowing correct tissue identity and transition from meriste-
matic to differentiated cells.
5.4 Conclusions
The molecular mechanisms controlling root architecture are being unraveled using
a variety of approaches combining physiology, genomics, and genetics. Major
questions remain to understand how these mechanisms interact with the soil stress
conditions, and the advent of genomic technologies may open new perspectives for
the analysis of how roots adapt to the soil environment. This work, mainly done in
model systems such as Arabidopsis and M. truncatula, uncover diverse regulatory
genes, notably TFs that participate in abiotic stress responses and genetic programs
regulating root growth and architecture. Integration of these data with genomic
approaches on different genetic backgrounds will reveal critical regulatory net-
works and molecular hubs, whose orthologs could then be analyzed in crop plants to
establish the generality of these mechanisms and impact agricultural practices.
Acknowledgments Laura de Lorenzo was supported by an F.P.U. (University Professor Training

grant) from the Spanish Department of Education and Science, Spain, and Ons Zahaf was the
recipient of a grant from the Tunisian Government, Tunis.
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Chapter 6
Mechanisms of Aluminum Tolerance
Owen A. Hoekenga and Jurandir V. Magalhaes
Contents
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
6.1.1 Scope of Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
6.1.2 Brief Overview of Al Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
6.2 Al Exclusion by Organic Acid Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
6.2.1 Mediated by Malate and ALMT1-Type Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
6.2.2 Mediated by Citrate and AltSB-Type Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
6.2.3 Mediated by Oxalate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
6.3 Al Exclusion by Non-organic Acid Dependent Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
6.3.1 Al Exclusion Mediated by Other Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
6.3.2 Mediated by pH Change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
6.4 Internal Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
6.4.1 Internal Chelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
6.4.2 Reactive Oxygen Species Scavenging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
6.4.3 Lipid Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
6.4.4 Cell Wall Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
6.5 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
O.A. Hoekenga (*)

Robert W. Holley Center for Agriculture and Health, US Department of Agriculture, Agricultural
Research Service, Ithaca, NY 14853, USA
e-mail: Owen.Hoekenga@ars.usda.gov
J.V. Magalhaes
Embrapa Maize and Sorghum, Rod. MG 424 Km 65, 35701-970 Sete Lagoas, Minas Gerais, Brazil
e-mail: jurandir@cnpms.embrapa.br

134 O.A. Hoekenga and J.V. Magalhaes
6.1 Introduction
6.1.1 Scope of Problem
The Food and Agriculture Organization (FAO) of the United Nations regards Al
toxicity as the second largest soil constraint to agriculture, after erosion hazard and
affects 14.7% of the world’s land area (Bot et al. 2000). In comparison, salinity and
sodicity each affects ~3% of the world’s land area. Al toxicity is the leading soil
constraint to agricultural production in Sub-Saharan Africa, Asia, Oceania, Central
and South America and the second largest limitation for North American agricul-
ture (Bot et al. 2000). Nearly one-third of the countries enumerated in a recent FAO
survey exhibit Al toxicity on 25% or more of their area (54/166 countries; Bot et al.
2000). Al toxicity exists at soil pH < 5.5, at which point rhizotoxic Al cations are
solubilized from non-toxic aluminosilicates and other minerals (Kochian 1995;
Kochian et al. 2004). While inhibition of root growth and function are early
consequences to Al intoxication, increased susceptibility to other stressors and
overall diminishment of yield are the latter consequences. For example, Brazil,
Argentina, and Colombia are the three largest maize producers in South America.
Brazil and Colombia have extensive land area with Al toxicity (63, 56%, respec-
tively) while Argentina has essentially no Al-intoxicated soils (Bot et al. 2000).
The 3-year (2004–2006) average maize yields in Colombia were one-third those
obtained in Argentina, while Brazil were approximately one-half (NASS 2007).
Without the Al stress limitation, significantly higher yields could be achieved on the
same arable land, which would promote food security, economic development, and
environmental preservation.
6.1.2 Brief Overview of Al Tolerance
Al rhizotoxicity occurs when Al cations reach vulnerable portions of the root,

without being detoxified. Al chelation is a common detoxification and can occur
outside (Al exclusion) or inside (internal chelation) the root. Al exclusion is the best
understood family of tolerance processes and may rely upon malate, citrate, or other
small molecules to chelate the Al. Several genes have been identified as major Al
tolerance genes in the malate and citrate pathways, while other Al exclusion path-
ways are less well defined. Al exclusion by chelation requires detection of Al,
synthesis and transport of ligands out of the root. The Al tolerance genes identified
to date fall into the third (transport) category. Al uptake into the root is nearly
unavoidable; plants have mechanisms for internal tolerance to Al stress. Internal
tolerance may result from intracellular chelation of Al, reactive oxygen species
(ROS) scavenging, modifications to lipid or cell wall synthesis, or other unknown
mechanisms.
6 Mechanisms of Aluminum Tolerance 135
Our goals for this review are to update recent progress in understanding the
molecular processes that underlie the mechanisms of Al tolerance. We have placed
emphasis on mechanisms where genes have been identified and confirmed to be
important for Al tolerance and devoted less space to the less clearly defined
mechanisms. We apologize in advance for any omission.
6.2 Al Exclusion by Organic Acid Release
6.2.1 Mediated by Malate and ALMT1-Type Transporters
6.2.1.1 Contributions from Wheat
Many discoveries in Al tolerance research were made in wheat, for both physiolog-
ical mechanisms and their underlying molecular components. The first Al tolerance
gene cloned from any species was the Al activated malate transporter (hereafter
referred to as TaALMT1) (Sasaki et al. 2004). This was accomplished via subtrac-
tive cDNA library sequencing performed on the ET8 and ES8 near isogenic lines,
which differ at the Alt1 locus found on the long arm of chromosome 4D (Delhaize
et al. 1993; Sasaki et al. 2004). TaALMT1 identifies a gene family with members in
Arabidopsis, rice, and many other plants (Sasaki et al. 2004). The most striking
polymorphism between wheat alleles is at the level of gene expression, where
the tolerant ET8 line had much higher levels of expression for TaALMT1 than
the sensitive ES8 (Sasaki et al. 2004). Biophysical analysis demonstrated that the
TaALMT1 protein responds to the presence of extracellular Al and is located within
the plasma membrane, consistent with the identification as an Al tolerance gene
(Sasaki et al. 2004; Yamaguchi et al. 2005).
Physiological analysis of a collection of wheat cultivars demonstrated that the
majority of differences in Al tolerance could be explained by the quantity of malate
released (Ryan et al. 1995). The strongly positive correlation suggested that genetic
differences in Al tolerance were concentrated within a single major tolerance
mechanism (r2 ¼ 0.84, malate efflux to relative root growth) (Ryan et al. 1995).
Subsequent molecular analyses of wheat germplasm collections have reinforced the
physiological observation; expression of the TaALMT1 gene is highly correlated
with both malate release and overall Al tolerance (Raman et al. 2005). Sequence
analysis of the TaALMT1 promoter region has revealed large structural differences
between tolerant and sensitive cultivars, with six clear haplotypes emerging within
cultivars that represent a wide range of Al tolerance (Sasaki et al. 2006; Raman
et al. 2007). These studies have reaffirmed the relationship between malate release
and Al tolerance, as estimated by relative root growth (r2 ¼ 0.88, 0.81 for Sasaki
et al. 2006 and Raman et al. 2007, respectively). The importance for any of the
motifs within the promoter haplotypes is not yet clear, but it has been hypothesized
that one or more motifs found in promoters with low expression have increased in
copy number and rearranged to derive stronger promoters (Delhaize et al. 2007).
This hypothesis is intriguing due to similarity with observations made at the AltSB
locus in Sorghum (see Sect. 6.2.2 below), but obviously requires additional experi-
mentation. While TaALMT1 expression is an important determinant for overall Al
tolerance, it is not the only one; gene expression differences explain one-half or less
of the differences in tolerance observed (Sasaki et al. 2006; Raman et al. 2007).
Multiple lines of genetic evidence support the observation that other factors
beyond TaALMT1 contribute to Al tolerance. First, analysis of the chromosomal
arm deletion stocks in the Chinese Spring background (ditelosomic chromosomes)
indicated that the loss of three different regions compromised Al tolerance
(Papernik et al. 2001). The loss of chromosome 4DL gave reduced root growth,
malate release, and increased Al accumulation in the root apex; this is easily
explained by the loss of TaALMT1 (Papernik et al. 2001). However, the loss of
the short arms of 5A and 7A also reduce Al tolerance by the same metrics, although
not as severely as losing 4DL (Papernik et al. 2001). Thus, at least three factors
contribute to Al-activated malate release in the Chinese Spring background. Sec-
ond, incomplete transfer of Al tolerance from Altas66 into a Chisholm background
(Chisholm-T) illustrates that multiple loci are important for the high degree of
tolerance observed in Atlas66 (Tang et al. 2002). Malate release in Chisholm-T was
approximately half that observed in Atlas66, where the Chisholm-T derivative
carries the Atlas66 allele of TaALMT1 (Tang et al. 2002; Guo et al. 2007a). The
Chisholm-T line has higher TaALMT1 expression than that seen in the sensitive
sister near isogenic line (Guo et al. 2007b). RT-PCR or other methods were not used
to make the direct comparison between Atlas66 and the Chisholm derivatives, and
so it is difficult to assess which degree cis-acting and trans-acting factors play to
determine TaALMT1 expression. However, it is clear that at least two loci are
important for determining the differences in tolerance observed between Atlas66
and Chisholm. Third, while TaALMT1 represents a major effect QTL in multiple
mapping populations, it does not explain all of the variance observed (Raman et al.
2005). Five doubled haploid populations were evaluated for Al tolerance; markers
within or tightly linked to TaALMT1 explained 75–93% of the variance in the trait
(Raman et al. 2005). Genome-wide marker scans were not conducted to locate the
other, minor QTL that contribute to the remainder of the genetic variance; the
authors mention the possible contributions from chromosomes 5AS and 7AS as
possible locations for minor QTL. However, the heritability of Al tolerance for
these mapping populations was not reported, the component of variance due to
genetic factors; it is possible that the heritability of Al tolerance is sufficiently low
that TaALMT1 explains all of the genetically determined differences in Al tolerance
by itself.
Other determinants for Al tolerance in wheat may include protein kinases or
phosphatases. Reversible protein phosphorylation is a common mechanism for
regulating protein activity and is known to be a point of control for many abiotic
stress responses, including salt, water, and cold stresses (Liu et al. 2000; Zhu 2001).
Malate release in wheat is Al activated, while TaALMT1 gene expression is not
(Sasaki et al. 2004; Raman et al. 2005). This indicates that much of the regulation
for malate release occurs at the protein level. Short pretreatment (30 min) of wheat
seedlings with the protein kinase inhibitors K-252a and staurosporine significantly
reduced malate efflux after Al challenge, while KN-62, calphostin C, and chelery-
thrine pretreatments had no effect (Osawa and Matsumoto 2001). Of the protein
phosphatase inhibitors tested, only okadaic acid had an effect. Okadaic acid and
staurosporine reduced malate release 30–40% while K-252a essentially abolished
malate release from treated seedlings (Osawa and Matsumoto 2001). Perhaps the
loci found on chromosomes 5AS or 7AS represent these pharmacologically sensi-
tive factors. It is clear that reversible phosphorylation plays a role in the perception
of Al, the first step in the Al-activated organic acid release pathway.
From a basic biology perspective, it is clear that Al tolerance research has made
great gains in wheat. From the applied biology perspective, two studies are espe-
cially noteworthy. First, as TaALMT1 represents a major Al tolerance QTL, having
genotypic information for this locus can allow marker-assisted breeding for Al
tolerance. This substitution of low-cost molecular genotyping for field-based phe-
notyping dramatically accelerates the pace of crop improvement. As a result of
germplasm surveys and the concomitant DNA sequence analyses, haplotype-specific
DNA markers have been generated for elite TaALMT1 alleles to support marker-
assisted breeding (Raman et al. 2007). This should permit the rapid movement of
highly tolerant alleles into elite varieties. Second, TaALMT1 can be utilized for
transgenic crop improvement purposes for species with little variation in Al toler-
ance. Barley is among the most Al-sensitive economically important cereals; while
variation does exist for Al tolerance between barley varieties, it does not provide
adequate protection against Al toxicity (Tang et al. 2000). The introduction of
TaALMT1 into barley resulted in dramatic enhancement of Al tolerance (Delhaize
et al. 2004). Where 2 mM Al concentrations inhibited root growth 50% for non-
transgenic controls and azygous sibling lines grown in hydroponic culture, 40 mM Al
was required to achieve the same level of inhibition for transgenic barley (Delhaize
et al. 2004). Similar results were also observed for soil-grown plants, although the
efficacy of these transgenic events is yet to be evaluated under field conditions.
6.2.1.2 Contributions from Arabidopsis
Arabidopsis thaliana does not possess a great degree of Al tolerance, unlike wheat
(Larsen et al. 1996). However, Arabidopsis is an excellent model system for
molecular genetic and physiological genomic analyses of Al tolerance. What
tolerance exists in Arabidopsis is largely due to Al-activated malate release and
both plants share at least one homologous protein (Hoekenga et al. 2003; Hoekenga
et al. 2006). The existence of a well-annotated and mutagenized genome with the
multitude of other genomics-based resources makes study in Arabidopsis highly
complementary to study in wheat.
TaALMT1 defines a gene family in Arabidopsis with more than a dozen
members (Hoekenga et al. 2006). Of these Arabidopsis, ALMT-like genes (hereafter
AtALMT), AtALMT8 is the most similar. The gene family has a diverse pattern of
gene expression according to publicly available gene expression databases, where

multiple AtALMT are expressed in essentially every tissue tested (Meyers et al.
2004; Kilian et al. 2007). However, mutant analysis indicates that only AtALMT1 is
essential for Al tolerance responses (Hoekenga et al. 2006). An AtALMT1 knockout
mutant lacks Al-activated malate release, but is capable of releasing malate under
low pH/phosphate deficiency stress conditions indicating that a second AtALMT
is likely active under those conditions (Hoekenga et al. 2006). A third locus,
AtALMT9, encodes a vacuolar malate transporter, expressed in nearly every cell
of the plant (Kovermann et al. 2007). AtALMT9 has a small biophysical response to
applied Al as measured when heterologous expressed in oocytes, suggesting that
related AtALMT proteins share multiple aspects of functionality (Kovermann et al.
2007). Thus, there is clear functional specialization for members of the AtALMT
family, even if the role for only two members has been identified.
Organic acid release in response to Al stress can be classified as immediate
(pattern I) or inducible (pattern II) (Ma et al. 2001). Wheat represents a pattern I
style organic acid release; this is consistent with the constitutive expression for the
TaALMT1 gene with responsiveness to Al coming at the protein level (Sasaki et al.
2004). Arabidopsis represents a pattern II style plant; this is consistent with the
AtALMT1 gene being strongly induced by Al stress, while also responding at
the protein level (Hoekenga et al. 2006). Protein phosphorylation is involved in
AtALMT1 regulation as it is for TaALMT1 (Kobayashi et al. 2007b). The protein
kinase inhibitor K-252a eliminates Al-activated malate release in Arabidopsis, much
like that seen in wheat. AtALMT1 gene expression is still enhanced by Al with
K-252a co-treatment, suggesting that this drug acts at the protein level to restrict
malate release (Kobayashi et al. 2007b). Unlike wheat, staurosporine (a kinase
inhibitor) and calyculin A (a phosphatase inhibitor) also reduce malate efflux in
Al-treated Arabidopsis; AtALMT1 gene expression does not increase with either of
these drugs, suggesting that reversible phosphorylation acts at both the transcrip-
tional and post-transcriptional level to regulate AtALMT1 (Kobayashi et al. 2007b).
Reversible phosphorylation is also important for inactivating AtALMT1. Reversal
experiments, moving plants from Al-containing media to Al-free solutions, indicate
that malate efflux can rapidly be inactivated in Arabidopsis (Kobayashi et al. 2007b).
Co-treatment with calyculin A prevents the inactivation of AtALMT1; malate release
rates remain high as in Al-treated plants (Kobayashi et al. 2007b). It would be
intriguing to see if TaALMT1 also requires protein phosphatases for inactivation of
transport function. No protein kinases or phosphatases are known to be involved in
AtALMT1 regulation. However, gene expression for WAK1, a wall-associated protein
kinase is rapidly induced (20 min) by Al treatment. Over-expression of this kinase
can also modestly increase Al tolerance in transgenic Arabidopsis, but a direct
connection to AtALMT1 is yet to be determined (Sivaguru et al. 2003).
Low pH stress and several other toxic metals can induce AtALMT1 gene expres-
sion to a small degree (5–20%) compared to Al. However, these treatments do
not activate AtALMT1, which speaks to the specificity of the Al stress response
(Kobayashi et al. 2007b). One can imagine commonality of rhizotoxicity between
Al and erbium or lanthanum; however, genetic analysis indicates that the tolerance
processes are distinct (Kobayashi et al. 2007a). Low pH and Al stress responses
have some degree of overlap, which is not unexpected as Al toxicity is largely
predicated by low pH. Proton and Al tolerance can be genetically and experimen-
tally separated (Ikka et al. 2007; Iuchi et al. 2007). This lack of concordance
between proton and Al stress tolerance was made several years ago in maize, and
the identification of STOP1 in Arabidopsis gives hints to the underlying molecular
mechanisms (Poschenrieder et al. 1995; Iuchi et al. 2007). STOP1 represents a
transcription factor required for proton stress tolerance responses; AtALMT1
expression requires the presence of STOP1 (Iuchi et al. 2007). The STOP1 null
mutant is hypersensitive to proton stress, but is also susceptible to Al toxicity at
doses that do not affect the growth of wild-type plants (Iuchi et al. 2007). It is not
yet clear whether STOP1 activates AtALMT1 transcription directly or indirectly
(e.g., acting at an earlier regulatory level), but this discovery is intriguing in the
light of the number of economically important plants that can be classified in
pattern II organic acid release. As ALMT1-like genes are shared between monocots
and dicots as essential Al tolerance genes, perhaps STOP1-like transcription factors
are also shared (Magalhaes 2006).
6.2.1.3 Contributions from Other Species
Al-activated malate release has been reported for species other than Arabidopsis
and wheat (Kochian et al. 2004). While many of the advancements in the area of
Al-activated malate release have been made in these species, several have not. Two
will be mentioned here. First, rapeseed (Brassica napus) has been reported on some
occasions to release both malate and citrate in response to Al stress (Zheng et al.
1998b). This appears to be cultivar-specific rather than a function of experimental
design. Dual organic acid release has also been reported in rye (Secale cereale),
cowpea (Vigna unguiculata), and soybean (Glycine max) (Li et al. 2000; Silva et al.
2001; Jemo et al. 2007). Rye is among the most Al tolerant of the cereals; perhaps
the dual release of organic acids contributes to its protection and can be exploited as
a target for plant improvement. The malate transporter important for Al tolerance
in rye has been identified as ScALMT1, while the citrate transporter is still unknown
(Fontecha et al. 2007). Given that malate and citrate transporters have both been
identified, presumably progress can be made in rye, cowpea, soybean, or other
species toward the goal of increasing Al tolerance through marker-assisted breeding
or biotechnology.
Second, increasing the numbers of organic acid transporters or interfering with
signal transduction pathways produces clear effects on organic acid release. Recall
that the process of organic acid release can be broken into three components:
perception of Al, synthesis of ligand, transport out of the root. The first and third
parts of this process can clearly be altered so as to affect Al tolerance. Manipulating
the second part of this process, organic acid synthesis, is much less reliable to alter
Al tolerance. Success has been reported in alfalfa (Medicago sativa) using malate
dehydrogenase and in rapeseed with citrate synthase (Tesfaye et al. 2001;
Anoop et al. 2003). However, over-expression of citrate synthase gave inconsistent

outcomes in transgenic Nicotiana (de la Fuente et al. 1997; Delhaize et al. 2001).
Organic acid supplies within the cell may or may not be limiting for effective stress
responses. In maize where organic acid release rates differed, no changes occurred
in organic acid pools that could be correlated with differential Al tolerance (Pineros
et al. 2005). In fact, the efficacy of over-expression of TaALMT1 in barley would
argue that organic acid supplies might not be a limitation to Al tolerance (Delhaize
et al. 2004). Perhaps with a more careful and systematic study of the interplay
between Al perception, ligand synthesis and release, patterns will emerge to better
instruct how these processes can be manipulated to improve Al stress tolerance.
6.2.2 Mediated by Citrate and AltSB-Type Transporters
6.2.2.1 Contributions from Sorghum
Several physiological mechanisms of Al tolerance have been proposed but the

agronomical efficacy to promote yield stability on acid soils remains at best
uncertain. The clear exception to this statement is the utility of Al-induced organic
acid release from root apices, which is certainly a major mechanism enabling
agriculture on acid, Al toxic soils. The improvement of barley with TaALMT1 by
transformation illustrates this, but only as a proof of concept (Delhaize et al. 2004).
A stronger example for the efficacy of improving Al tolerance in crop plants is the
discovery and characterization of the major Al tolerance gene in Sorghum, AltSB
(Magalhaes et al. 2007). The cultivar SC283 is the best known Al tolerance
standard in Sorghum and has repeatedly shown superior agronomic performance
on acid soils (Duncan et al. 1983; Duncan 1988). Subsequently, using hydroponic
culture rather than field-based observations, Al tolerance in cv. SC283 was shown
to be largely under the control of a single, semi-dominant gene, AltSB, which was
mapped to the end region of Sorghum chromosome 3 (Magalhaes et al. 2004). This
gene was identified by map-based cloning and shown to underlie Al-induced citrate
release, the primary Al tolerance mechanism at work in Sorghum (Magalhaes et al.
2007). The fact that segregation of AltSB was sufficient to explain ~80% of the
phenotypic variation for root growth inhibition caused by Al in hydroponic culture
strongly suggests that AltSB is also the major determinant for the superior agrono-
mical performance displayed by SC283 on Al-intoxicated acid soils. It should be
noted, however, that Al toxicity is one of the most important but not the only source
of abiotic stress on acid soils. Therefore, other genes related to adaptation to the
“acid soil syndrome” should also be considered. Nevertheless, recent comparisons
for agronomic performance on acid soils and root growth inhibition in hydroponic
culture indicated that the two traits are highly correlated in Sorghum; genotypes
carrying the AltSB allele from SC283 out-produced sister lines with an inferior AltSB
allele by ~1 mt ha-1 (Magalhaes et al., unpublished). The AltSB gene encodes a
member of the Multidrug and Toxic Compound Extrusion (MATE) family; the
gene is Al inducible with maximal levels of expression after several days of Al

stress (Magalhaes et al. 2007). In the original mapping population, the AltSB alleles
contained no polymorphisms within the protein coding sequence; rather, significant
differences were observed in the promoter region. A MITE-class transposon and
sequences immediately flanking it created a repeat unit of 243 bp; the sensitive
allele contained three repetitions while the tolerant (SC283) allele contained five
(Magalhaes et al. 2007). The number of repetitions is positively correlated with
citrate release, root growth, and gene expression. A relatively high level of consti-
tutive expression of AltSB in a tolerant isogenic line was not accompanied by large
and rapid citrate efflux. This suggests the regulation of the gene and the activity of
the protein are somewhat more complicated than the typical; perhaps, Al is required
to activate transport activity or gene expression does not occur in the epidermis in
the absence of Al. Experiments are underway to answer these questions.
6.2.2.2 Contributions from Barley
A locus controlling Al tolerance, Alp, was located to chromosome 4H by trisomic

analysis (Minella and Sorrells 1997). Subsequently, Alp was mapped to the long
arm of barley chromosome 4H in a population derived from the cultivar Dayton,
and subsequent studies using different mapping populations also identified Al
tolerance gene(s) the same chromosome (Tang et al. 2000; Raman et al. 2002;
Ma et al. 2004b). In a broader survey with 21 barley varieties, citrate release and Al
tolerance were positively correlated, while citrate release and Al content in root
apices were negatively correlated, indicating that Al exclusion mediated by citrate
was responsible for Al tolerance in barley (Zhao et al. 2003). This conclusion was
confirmed and expanded by Ma et al. (2004b), who reported co-localization
between the Al tolerance gene on chromosome 4H and rates of citrate release.
Complete linkage of a barley homolog of the MATE family (HvMATE) with the Alp
locus was reported in a doubled haploid population (Wang et al. 2007). Expression
of HvMATE was also correlated to Al tolerance and Al-activated citrate efflux,
leading the authors to consider the hypothesis that HvMATE underlies the Alp locus
in barley. Fine-scale genetic mapping and microarray analysis confirmed that a
member of the MATE family, HvAACT1, confers Al tolerance in barley (Furukawa
et al. 2007). The HvAACT1 gene cloned by Furukawa and co-workers enhanced
Al-activated citrate release and Al tolerance in transgenic tobacco. The protein was
localized to the epidermal cells of barley root tips and within the plasma membrane,
according to GFP translational fusions. In addition, heterologous expression of
HvAACT1 in Xenopus laevis oocytes indicated that the protein was permeable to
citrate rather than malate (Furukawa et al. 2007).
Sorghum and barley have several similarities for the inheritance of Al tolerance;
both display rather simple genetic control for Al tolerance and rely on homologous
genes. However, barley is considered to be the most sensitive species among the
cereals, whereas some Sorghum accessions may exhibit extremely high levels of Al
tolerance (Wang et al. 2006). A comparison between HvMATE and AltSB protein
sequences uncovers several significant differences, as they are only 65% identical
and 79% similar. Also, they possess strikingly different features such as exon/intron
structure in addition to apparently different numbers of putative transmembrane
domains. Although similarities do exist between the two genes, such as a level of
constitutive expression in the absence of Al and a likely Al activation of the Sorghum
and barley MATE proteins, structural differences may account for the remarkably
different levels of Al tolerance encoded by HvMATE and AltSB. A third related
MATE transporter, FRD3 from Arabidopsis, could also contribute to Al exclusion
via citrate release (Durrett et al. 2007). Ectopic expression of FRD3, which is
normally involved with iron metabolism and transport, is capable of making a modest
increase to Arabidopsis Al tolerance (Durrett et al. 2007). Comparative analysis
between the three MATE proteins, AltSB, HvMATE, and FRD3, will likely reveal
domains and residues important for citrate transport and Al activation.
6.2.2.3 Contributions from Rye
Unlike barley, rye is one of the most Al-tolerant cereals (Aniol and Gustafson
1984). In part, this may result from additive effects of malate and citrate, which are
both released when some rye genotypes are exposed to Al (Li et al. 2000). Studies
with Triticale, which is a hybrid between wheat and rye, identified that gene(s) on
the short arm of rye 3R are required for organic acid release in Triticale (Ma et al.
2000). The pattern of organic acid release in rye involves a lag phase after the
addition of Al (pattern II), suggesting induction at the gene or protein level to
convey full activity. Interestingly, citrate release as modulated by the Sorghum Al
tolerance gene AltSB is also inducible over time of exposure to Al, a response that is
paralleled by AltSB expression (Magalhaes et al. 2007). Given that rye chromosome
3R is homoeologous to Sorghum chromosome 3, it is possible that a MATE
ortholog of AltSB is responsible for rye citrate release (Magalhaes et al. 2004).
6.2.3 Mediated by Oxalate
Malate and citrate are not the only organic acid ligands for Al reported in root
exudates. Oxalate has also been reported to appear in root exudates from Al-treated
plants and is an effective chelate, intermediate between citrate and malate in terms
of the dissociation constant for Al binding. Al-activated oxalate release has been
reported in buckwheat (Fagopyrum esculentum), maize, taro (Colocasia escul-
tenta), and alfalfa (Medicago sativa) (Ma and Miyasaka 1998; Zheng et al.
1998a; Kidd et al. 2001; Tesfaye et al. 2001). Oxalate is only mentioned in passing
in this review due to the lack of identification for an Al-activated oxalate trans-
porter. It is possible that ALMT1-type or AltSB-type transporters are permeable to
oxalate in addition to malate and citrate, respectively. However, the oxalate trans-
porter may represent a third class of organic acid transporters and is yet to be
discovered and described.
6.3 Al Exclusion by Non-organic Acid Dependent Mechanisms
Al tolerance is highly correlated with exclusion of Al from the root apex in many
species. Al exclusion explained the majority of differences in root growth observed
between a small panel of maize varieties (Pineros et al. 2005). However, low and
high outliers caused Piñeros and co-workers (2005) to reject the hypothesis that all
Al exclusion in maize is mediated by organic acid release. Exclusion could also
result from chelation by non-organic acid ligands or increasing rhizosphere pH,
which would change the speciation of Al to less or non-toxic forms. Organic acid
release does not explain the high degree of tolerance observed in rice or Brachiaria
decumbens (Wenzl et al. 2001; Ma et al. 2002). Thus, it is likely that other species
will be similar to maize, where Al tolerance is dependent upon multiple, indepen-
dent mechanisms.
6.3.1 Al Exclusion Mediated by Other Ligands
Evidence for Al exclusion mediated by non-organic acid ligands is relatively

limited. This may be in part due to the difficulties in detection and identification
of root exudates. Presumably, with the advancements in non-targeted metabolomic
analysis via mass spectrometry or nuclear magnetic resonance, comprehensive
analysis of root exudates will be more common in the future (see (Keurentjes
et al. 2006) for example of this methodology).
Beyond organic acids, two classes of compounds have been implicated in Al
tolerance. First, inorganic phosphate release was reported to occur concomitantly
with malate in wheat (Pellet et al. 1996). Phosphate has high affinity for Al and
would therefore make an effective ligand, although an expensive one from a
nutritional standpoint. In this wheat study, a constitutive phosphate release was
observed in the Al tolerant variety tested that was largely absent from the sensitive
variety and from near isogenic derivatives with differing levels of tolerance (Pellet
et al. 1996). This suggested that the same transporter did not mediate the release of
malate and phosphate. Phosphate, like malate, release was spatially restricted to the
root apex; however, the contribution of phosphate release to overall Al tolerance
was unclear (Pellet et al. 1996). Phosphate has also been reported to occur in
Arabidopsis. Like wheat, the Al tolerant variety released more phosphate and
again the release was not Al responsive (Hoekenga et al. 2003). In the Arabidopsis
case, essentially all the differences observed between varieties for Al tolerance
were consistent with the differences in malate release, such that it is unclear
whether the phosphate release observed made a contribution (Hoekenga et al.
2003). Further analysis will be required to evaluate the importance of phosphate
release to Al tolerance relative to it being just coincidental.
Second, phenolic compounds have been implicated in Al exclusion. Both flava-
noid phenolics and oxalic acid were observed in root exudates from Al-treated
maize seedlings (Kidd et al. 2001). Similar patterns of oxalate release were
observed in three different maize cultivars with varying levels of Al tolerance,
suggesting that oxalate release did not correlate with the differences observed in Al
tolerance. However, different patterns of catechol, catechin, curcumin, and querce-
tin release were observed between the three maize varieties, with the magnitude of
the catechin release most concordant with the Al tolerance differences (Kidd et al.
2001). Catechin is structurally similar to morin, which is commonly used as an
Al-binding dye and means to assess Al absorption (Eggert 1970). Both catechin and
morin have high affinity for binding with Al, meeting or exceeding the affinity
observed for Al-organic acid complexes. Catechin exudation may represent an Al
tolerance mechanism, but validation of this hypothesis requires more evidence.
6.3.2 Mediated by pH Change
Al speciation is pH dependent, with the different cations (e.g., Al3+, Al(OH)2+)

exhibiting different levels of rhizotoxicity (Kinraide 1991). Relatively small differ-
ences in rhizosphere pH can shift the balance from a preponderance of highly
rhizotoxic Al3+ to the less toxic hydroxy-Al compounds. The first evidence that pH
gradients at the root surface could confer Al tolerance came in Arabidopsis with the
identification of a mutant, alr-104 (Degenhardt et al. 1998). An increased rate of
proton influx was observed in the mutant, which led to an alkalinization of the
rhizosphere by ~0.15 pH units (Degenhardt et al. 1998). Buffering the pH of the
nutrient solution abolished the increased level of Al tolerance. A second, similar
mutation was reported in Arabidopsis in the form of the alt1 locus (Gabrielson et al.
2006). While pH buffering of the nutrient solution abolished the increase in Al
tolerance observed with alt1, rhizosphere pH was not mapped and thus is difficult
to compare the two studies directly (Gabrielson et al. 2006). Root surface pH has
been measured in maize, to examine whether pH gradients might contribute to Al
tolerance differences (Pineros et al. 2005). Differences in root tip pH were observed
between varieties in the absence of Al treatment, with the most tolerant variety
possessing the most alkaline pH. However, Al treatment collapsed any differences
observed along the root surface between varieties and were not restored within 72 h
(Pineros et al. 2005). It is still possible that root surface pH differences do contribute
to natural variation in Al tolerance, but the proper study system is yet to be identified.
6.4 Internal Tolerance
Uncontrolled uptake of Al into the root is essentially inevitable, despite the best
efforts of the various Al ligands. Internal tolerance to Al stress is therefore impor-
tant to some greater or lesser degree for all plant species. In fact, the most highly
Al-tolerant species largely or exclusively rely on internal tolerance mechanisms.
For a review on the mechanisms of Al hyperaccumulation, the reader is directed to

Watanabe and Osaki (2002). Some internal and external tolerance processes share
underlying physiological processes (e.g., chelation) while others are distinct.
6.4.1 Internal Chelation
Organic acids are an important source for internal as well as external Al tolerance.
Many highly Al-tolerant species, including those considered to be Al hyperaccumu-
lators, utilize organic acid chelation within the root or shoot to achieve Al tolerance.
Buckwheat has been reported to use oxalate for both external and internal chelation
of Al (Zheng et al. 1998a; Ma and Hiradate 2000; Shen et al. 2002). Oxalate is also
the predominant intracellular ligand in tea (Camellia sinensis) roots (Morita et al.
2008). On the other hand, citrate is the predominant ligand found in xylem sap, to
promote the long distance transport of Al from the root to shoot (Morita et al. 2004).
Internal organic acid concentrations respond to Al treatment in Brachiaria roots
(Wenzl et al. 2002). Organic acid levels increase several fold in the whole root for
both tolerant Brachiaria decumbens and sensitive Brachiaria ruziziensis, where most
of the organic acids are concentrated in the root apices. While the tolerant accessions
accumulate more than the sensitive ones, the difference is far too small to explain the
dramatic differences in Al tolerance (Wenzl et al. 2002). Similarly, citrate content
increased in maize root apices due to Al treatment; however, concentrations were
equivalent among the six maize inbreds and thus not correlated with Al tolerance
(Pineros et al. 2005). It is important to note, however, in spite of the fact that internal
organic acid concentrations may not correlate with differences observed in Al
tolerance between Brachiaria and maize accessions; this does not exclude the
possible importance for internal organic acid chelation. Rather, it may be that internal
organic acid chelation is essential for Al tolerance but not genetically variable, at
least within the accessions that have been studied to date.
Phenolic ligands are often used for long-term storage of Al in cells of hyper-
accumulator species. While organic acids are used to transport Al within tea,
catechin is the predominant ligand for Al sequestration in tea leaves (Nagata
et al. 1992). Delphinidin and chlorogenic acid are associated with Al in Hydrangea
sepals; association of these pigments with Al influences flower color (Takeda et al.
1990). Delphinidin is an anthocyanin, while chlorogenic acid is a phenylpropanoid;
both are related to catechin, a flavanoid. Each of these Al ligands represents
separate branches of a phenolic family tree, where early biosynthetic reactions
are shared. For example, chalcone synthase (CHS) is the committing step for the
synthesis of catechin, chlorogenic acid, and delphinidin. In maize, variation at
chalcone synthase loci is significant for resistance to insect herbivores (Szalma
et al. 2002). Anthocyanins are induced by abiotic stresses such as cold and high
light (Christie et al. 1994; Kimura et al. 2003). Phenylalanine ammonia lyase
(PAL), a key gene in primary metabolism, carries out the biosynthetic step prior
to CHS and is known to be an Al-inducible gene (Snowden and Gardner 1993).
While some have attributed the induction of PAL and CHS by Al treatment as non-
specific stress responses, it is also possible that these changes in gene expression
underlie Al tolerance processes dependent upon phenolic ligands. As systems
biology approaches are applied to the study of Al tolerance, it should be increas-
ingly possible to identify which stress responses due convey Al tolerance over the
noise of non-specific changes in gene, protein, or metabolite expression (Hoekenga
et al. 2003, 2006; Yang et al. 2007; Zhang et al. 2007).
Hydroxyamates are another class compounds with potential importance to Al
tolerance. Perhaps the best known hydroxyamate is 2,4-dihydroxy-7-methoxy-1,
4-benzoxazin-3-one (DIMBOA) (Frey et al. 1997). DIMBOA is highly effective at
controlling insect herbivores and microbial pathogens; the complete synthetic
pathway was recently determined (Jonczyk et al. 2008). DIMBOA has also been
implicated in other biological processes, including auxin-induced elongation of
maize coleoptiles (Park et al. 2001). Poschenrieder and colleagues, who also were
the first to make the Al-flavanoid connection in maize, demonstrated intracellular
Al tolerance due to DIMBOA–chelation of Al (Poschenrieder et al. 2005). Hydro-
xyamates are also found in nature as siderophores, ligands used by bacteria to
acquire essential metals from the soil solution or to protect against toxins.
A siderophore-deficient mutant strain of Bacillus has long been known to be
sensitive to Al stress (Davis et al. 1971). Al stress elicited siderophore exudation
from wild-type Bacillus cells, which tolerated Al treatments that completely inhib-
ited growth in the siderophore mutant (Davis et al. 1971). Media supplementation
with the Bacillus hydroxyamate siderophore, schizokinen, or the Rhizobium side-
rophore, vicibactin, conferred tolerance to Al stress to those species, respectively
(Davis et al. 1971; Rogers 1986). As with the phenolic ligands, the application of
systems biology approaches to Al stress tolerance will likely demonstrate the
efficacy of hydroxyamate and others as contributors to Al tolerance processes
across multiple species, genera, and wider evolutionary relationships.
6.4.2 Reactive Oxygen Species Scavenging
Al stress generates ROS, like many other abiotic stressors (Cakmak and Horst
1991). Whether these ROS are a primary or secondary effect of Al toxicity is
arguable; however, the damage done to lipids, nucleic acids, and other susceptible
molecules is not (Yamamoto et al. 2001). ROS-responsive genes have been
detected by gene and protein expression profiling methods in multiple species
(Richards et al. 1998; Yang et al. 2007; Zhang et al. 2007). Genetic analysis has
not implicated ROS scavenging genes, or their regulators, as responsible for natural
variation in Al tolerance. Transgenic experiments that overexpress superoxide
dismutase, peroxidase, or glutathione S-transferase do increase Al tolerance by
small but significant degrees (Ezaki et al. 2000; Basu et al. 2001). It is certain that
ROS scavenging contributes to internal Al tolerance and may be especially impor-
tant in plants that do not rely upon Al exclusion.
6.4.3 Lipid Composition
Lipid peroxidation is an early sign of damage to Al-intoxicated roots (Yamamoto

et al. 2001). The degree of lipid peroxidation is variable between Al-tolerant and -
sensitive accessions, but it is unclear whether these differences are to due the
proximate or ultimate causes of Al tolerance. One can imagine either scenario:
(1) lipid composition is variable, thus making some plant less susceptible to
peroxidation (a proximate cause of tolerance) or (2) plants with highly effective
Al exclusion mechanisms suffer less lipid peroxidation, as less Al3+ reaches the
plasma membrane (an ultimate cause). A wheat phosphatidylserine synthase gene
was capable of increasing Al tolerance in the yeast, S. cerevisiae (Delhaize et al.
1999). The transgene dramatically reduced phosphatidylinositol levels while
increasing phosphatidylserine, which presumably reduced Al/ROS susceptibility
(Delhaize et al. 1999). The result from yeast was not reproduced in plants, as each
has specific requirements for functional membranes (Delhaize et al. 1999). This did
stimulate the examination of lipid composition between Al-tolerant and -sensitive
varieties (Chaffai et al. 2005; da Silva et al. 2006). Lipid profiling in maize root tips
suggested that sphingolipid composition might be correlated with Al tolerance;
subsequent transgenic experiments with a D8 sphingolipid desaturase in maize,
Arabidopsis and yeast verified this hypothesis (da Silva et al. 2006; Ryan et al.
2007). Together, the lipid profiling and transgenic experiments indicate that lipid
composition can be a proximate cause of Al tolerance.
6.4.4 Cell Wall Composition
The majority of Al associated with the root (80%) can be found in the cell wall,
according to estimates from maize and wheat (Ma et al. 2004a; Wang et al. 2004).
This association presumably accounts for the reduction in wall extensibility
observed with Al-treated plants (Jones et al. 2006; Zakir Hossain et al. 2006).
Differences between tolerant and sensitive accessions beg the proximate/ultimate
causes question: do tolerant accessions construct cell walls significantly differently
than sensitive accessions, or are the differences observed merely due to Al exclu-
sion. Cell wall composition does change in response to Al treatment, especially in
the pectin component (Eticha et al. 2005; Zakir Hossain et al. 2006; Yang et al.
2008). This is potentially significant as de-esterification of pectin increases the
density of negative charge within the cell wall; increasing the net negative charge
could allow greater Al loading onto the cell wall (Cosgrove 2005). Increasing the
esterified fraction of pectins has been correlated with increasing cell elongation
rates in Arabidopsis (Derbyshire et al. 2007). In both maize and wheat, Al tolerant
accessions had higher degrees of pectin methyl-esterification and reported lower
uptake of Al into the cell wall (Eticha et al. 2005; Yang et al. 2008). Unfortunately,
both studies were comparisons between a pair of accessions, one tolerant and one
sensitive. The statistical power for such a comparison is very low, but still the
results are intriguing. Additionally, Al3+ is a potent inhibitor of expansins, the
family of cell wall loosening enzymes responsible for acid-responsive growth
(Cosgrove 2000). Cell wall loosening and elongation is diminished or eliminated
in the absence of expansin activity; if Al3+ inactivates expansins, this could
also explain the rapid loss of root growth observed in Al-intoxicated roots.
If Al-resistant expansin isoforms exist, they could represent very powerful Al
tolerance loci as they could protect cell elongation in the presence of stress.
6.5 Concluding Remarks
It is an exciting time to be working in the Al tolerance field. Al tolerance genes have

been identified that underlie major QTL of agronomic importance. Given the size
and strength of the Al tolerance community, we anticipate many more discoveries
of similar magnitude in the coming years. Systems biology approaches that lever-
age traditional plant physiology against genome sequences and other technologies
will permit large improvement in Al tolerance. This should produce outcomes that
promote food security, economic development, and environmental protection in
acid soil regions.
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Chapter 7
Root Responses to Major Abiotic Stresses
in Flooded Soils
Rogerio O. Sousa and Antonio Costa de Oliveira
Contents
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
7.2 Iron in Flooded Soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
7.2.1 Iron Toxicity Symptoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
7.2.2 Iron Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
7.2.3 Iron Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
7.2.4 Iron Transport and Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
7.2.5 Improving high/low Iron Tolerance in Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
7.2.6 Mutation Inducing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
7.3 Toxicity of Organic Acids to Irrigated Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
7.3.1 Organic Acid Genesis in Flooded Soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
7.3.2 Organic Acid Toxicity Symptoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
7.4 Conclusion and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
7.1 Introduction
Soil flooding alters the natural equilibrium of components and organic matter
decomposition, unchaining a series of transformations that affect chemical and
physical soil attributes. Such changes are beneficial to the rice crop because rice
plants present morphological and molecular adaptations in order to survive these
environments that lack free molecular oxygen; moreover, most of nutrients increase
their availability in flooded conditions (Sousa et al. 2009). However, the soil
changes associated to flooding can result in stresses even to the rice crop, which is
well adapted to these conditions. These changes generate products such as soluble
R.O. Sousa (*)

Department of soils, Eliseu Maciel School of Agronomy, Federal University of Pelotas, Campus
UFPel, Capão do Leão RS-96001-970, Brazil
e-mail: rogerio.sousa@pq.cnpq.br
A. Costa de Oliveira
Plant Genomics and Breeding Center, Eliseu Maciel School of Agronomy, Federal University of
Pelotas, Campus UFPel, Capão do Leão -RS-96001-970, Brazil
e-mail: antonio.oliveira@pq.cnpq.br

156 R.O. Sousa and A. Costa de Oliveira
iron and short-chain organic acids which, under proper conditions, can be toxic to
rice. In order to achieve a perfect state of growth, the plant must balance the presence
of minerals at different concentrations and equate its needs. Among the major
stresses faced by rice plants under no tillage cropping systems in South America,
iron and organic acid toxicity top the list and will be the focus of this review.
Iron toxicity in the rice crop is a nutritional disorder that occurs in cultivated
fields of many countries, mainly in Asia, Africa, and South America. This disorder
has been named differently according to each country, symptoms, and occurrence
conditions. In Japan, for example, it is denominated “Akagare” type I, in Ceylon and
India, it is known as “Bronzing,” and in Colombia, it is known as “Anaranjamiento.”
In Brazil, iron toxicity has already been observed in many rice production areas,
especially from the introduction of modern-type cultivars, which occurred in the
middle of 1980s (Vieira et al. 1999). Organic acid toxicity is a stress that became
important after no tillage cropping started and increasing amounts of organic matter
originating from the straw of the previous crop started to take part in the process.
7.2 Iron in Flooded Soils
Iron is one of the most abundant elements on earth, contributing to approximately

5% of its total weight (Murad and Fischer 1988), and it is present in all soils, in
amounts ranging from 0.7 to 55% (Lindsay 1979). In the soil, iron oxides can be
uniformly distributed or concentrated in some profile layers, forming mottles,
nodules, concretions, hardpans, plinthites ou laterites. The main forms of iron
oxides in soils are hematite, goethite, lepidocrocite, and ferrihydrite, although
other oxide/hydroxides may be present (Sousa et al. 2004).
The changes in oxide and reduction states that occur in environments that
alternate between dry and flooded conditions, such as lowland soils cultivated
with rice, are determinant to the iron oxide and hydroxide forms that predominate
(Moormann and Van Breemen 1978). During flooding, a part of the soluble Fe2+
ions, which are rapidly oxidized during the following draining period, are precipi-
tated as ill-crystalized Fe3+ oxides. If the draining period persists, the Fe3+ oxide
degree of crystalization can increase, although in a very slow process. When the
draining period is over, Fe3+ oxides are again reduced and solubilized. The alterna-
tion between flooded and non-flooded conditions therefore favors low crystalinity
iron oxides. Thus, goethite, lepidocrocite, and ferrihidrite are the most common
forms of iron oxides in hydromorphic soils (Allen and Hajek 1989; Schwertmann
and Taylor 1989). According to van Breemen (1988), iron can still be present in
“green-rust” (Fe3+ and Fe2+ associated to Cl-, SO42 and CO32 anions in the
interlayers), siderite, pirite, and silicate minerals, such as smectites.
Soil bacteria that reduce Fe3+ in the soil have preference for the ill-crystalized
iron oxide forms. Thus, the speed and the amount in which iron oxides are reduced
and released to soil solution depend, among other factors, from the ratio of crystal-
ized and ill-crystalized soil minerals. As a consequence, the lower the degree of iron
7 Root Responses to Major Abiotic Stresses in Flooded Soils 157
oxide crystalinity, the higher the iron reduction and release into the soil solution
(Sousa et al. 2004). However, since the reduction of iron depends on the biological
activity, other factors must be considered in this process, as organic matter content
and the presence of easily reducible compounds, such as nitrate and manganese
oxides. Better fitted regression models were obtained for the prediction of exchange-
able Fe2+ (Sousa et al. 2004), during the flooding of 32 hydromorphic soils,
considering not only the amount of iron oxide, but also the amounts of Mn (extracted
with ammonium oxalate at pH 6.0), NO3 and organic carbon (Table 7.1).
In Fig. 7.1, the trend of iron concentrations in solution of two flooded soils, a
Planossolo (typic Albaqualf, USDA soil taxonomy) which can present iron toxicity
Table 7.1 Regression fitting models for exchangeable Fe2+ and NO3,
organic-C, and iron and manganese oxides soluble in ammonium oxalate
Model r2
Fe ¼ 3.82 + 0.061 Feo
2+
00.17b
Fe2+ ¼ 1.61 + 0.50 Feoa 00.50b
Fe ¼ 2.39 + 0.51 Feo – 0.30 Mno
2+ a a
00.54b
Fe2+ ¼ 3.78 + 0.59 Feoa – 0.37 Mnoa – 0.07 NO3 00.59b
Fe ¼ 4.38 + 0.52 Feo – 0.29 Mno – 0.11 NO3 + 0.42 C
2+ a a
00.64b
Source: Vahl (personal communication)
Feo – extracted with ammonium oxalate at pH 3
a
Feo and Mno – extracted with ammonium oxalate at pH 6
b
significant at 1%
250
Albaqualf
Chernosol
200
Fe2+, mg L–1
150
100
50
0
0 1 2 3 4 5 6 7 8 9 10 11 12
Weeks of flooding
Fig. 7.1 Iron contents in soil solution from two lowland soils as a function of flooding period.
Albaqualf: O.M: ¼ 17g kg1 ; Feoxalate ¼ 1:4g kg1 Chernosol: O.M: ¼ 24g kg1 ;
1
Feoxalate ¼ 0:4g kg
Source: Adapted from Sousa et al. (2009)
and a Mollisol where this nutritional disorder is not observed. In the Planossolo, in 4
or 5 weeks of flooding, iron concentration peeks high enough and toxicity to plants
can be reached. In this soil, the iron concentration peeks normally occur in the stage
where rice is most sensitive, which is the end of tillering. In Mollisol, the iron
amounts released to the soil solution are lower since this soil has low active iron
content (iron extracted with ammonium oxalate), and normally does not present
toxicity problems.
Iron toxicity in irrigated rice is commonly associated to some soil traits, such as
low pH, high iron oxide content, and low CEC. However, it is common to observe iron
toxicity symptoms at different pH, iron oxide contents, and CEC conditions (Sousa
et al. 2004).The first idea that one grasps about the toxicity of an element to plants is
that high concentrations of this element in the soil lead to an excess absorption and
toxicity. However, in iron toxicity, this idea cannot be taken as a common rule, since
there have been reports of symptoms occurring in crops growing in low iron soils and
no symptoms in crops growing in high iron content soils (Sousa et al. 2004).
The low soil pH is pointed as one factor that favors iron toxicity occurrence. In
this condition, iron solubility is higher, soil CEC is lower, and CEC saturation by
(H+ + Al3+) is higher. However, reports have described soil samples collected from
rice fields showing toxicity symptoms with pH values ranging from 3.8 to 7.5
(Sousa et al. 2004). The high soil iron content, low pH, and low CEC cannot be
considered, alone, as obligatory conditions for iron toxicity to occur, since many
reports have shown rice fields developing iron toxicity symptoms and showing
different iron content, pH, and CEC values. The detection of symptoms depends on
different soil and plant attributes, related to toxicity. A soil with high iron content,
but high CEC and base saturation, can present high Fe2+ content as a consequence
of flooding, but this can be low when compared to other cations such as K, Ca, and
Mg, as a result of CEC and base saturation values, resulting in healthy plants.
Another soil with low iron content, but low CEC, can present low amounts of Fe2+
during flooding, but, however, due to the low CEC, the ratio Fe2+/other cations can
be higher and consequently reach levels toxic to rice plants (Sousa et al. 2004).
Some unpublished results do exist for iron toxicity occurrence in land-leveled
areas. The preparation of these areas for rice cropping can give rise to iron toxicity
cases due to two factors: exposition of B horizon with higher iron contents, or
exposition of E layer, rich in sand and with lower ability to supply nutrients to the
plants (Sousa et al. 2004).
7.2.1 Iron Toxicity Symptoms
Iron toxicity is visually divided into two major symptom groups (Fig. 7.2): direct
toxicity or bronzing and indirect toxicity or yellowing. Direct toxicity is caused by
excessive iron absorption, while indirect toxicity is associated to overall nutrient
deficiency, induced by high iron content in the soil solution. These terms have been
adopted by the majority of authors in order to define the major iron toxicity-related
Fig. 7.2 Regular symptoms of iron toxicity. (a) and (b) Indirect toxicity; (c) direct toxicity; (d)
direct and indirect toxicity simultaneously.
symptoms. However, Sahrawat (2004) proposed recently the idea of induced

toxicity or fake toxicity, when the symptoms are caused by a multiple nutrient
deficiency (indirect toxicity) and true toxicity when symptoms are the result of high
iron content (direct toxicity).
The symptoms attributed to direct toxicity are composed of many dark brown
spots, which initiate in the tips and spread to the base of older leaves (Mengel and
Kirkby 1987). Similar symptoms were described in which, at higher iron contents,
the dark brown spots fuse, forming large dark brown areas in the leaves (Tanaka
et al. 1966). These points match the high iron concentration spots in the leaf. Similar
bronzing symptoms have been described (Mengel and Kirkby 1987; Bienfait 1985).
Also, it was reported that when the disorder progresses, leaves senesce and die, and
more severely injured plants show lower tillering, smaller panicles with high
percentage of sterile spikelets and lesser branched roots, with dark brown color
(Ponnamperuma et al. 1955; Sousa et al. 2004). Although the degree of toxicity
measures has been based on the degree of bronzing (IRRI 1965; Ota 1968), the
phenotype itself and the basis of tolerance are not well understood (Ota 1968; Peng
and Yamauchi 1993; Briat and Lobréaux 1997).
Indirect toxicity symptoms initiate with a yellowing of older leaf tips, which
evolves toward the base. Subsequently, the younger leaves are also affected and
many lower leaves die. In severe cases, leaves acquire an orange or yellow color
and may present dark brown stripes (Howeler 1973; Ottow et al. 1983).
Some authors make no distinction between direct or indirect toxicity, describing
the symptoms in the following way: iron toxicity is characterized by the develop-
ment of very small spots in older leaves, which gradually coalesce, giving a purple,
brownish red, orange, or yellow color spreading to the leaf base, especially on the
edges. These parts, then, become dry and curly toward the center. During the first
stages, younger leaves and the unaffected parts of older leaves are green, but later,
younger leaves also tend to show small dark brown spots, while older leaves dry
completely giving the plant a burned look. The root system is dark brown, thick,
and scarce; plant growth is stunted; and there is a high percentage of sterile flowers
(Lantin and Neue 1988).
In case the toxicity occurs during the plantlet stage, rice plants remain stunted
with a very limited tillering ability (Abraham and Pandey 1989). Toxicity during the
vegetative stage is associated with the reduction of plant height and dry matter
accumulation (Abu et al. 1989), which is greatly affected by root biomass (Fageria
1988). Tiller formation and number of fertile tillers can be severely reduced
(Cheema et al. 1990). When iron toxicity occurs at the end of the vegetative phase
or at the reproductive phase, the number of panicles formed decreases (Singh et al.
1992), there is an increase in spikelet sterility (Virmani 1977) and a delay in
flowering and maturation. In highly susceptible cultivars, flowering may not occur
(Ayotade 1979). Also, root growth can stop and the aerenchyma can senesce and
decay, resulting in a decrease of root oxidation ability and formation of Fe(OH)3
compounds on root surface changing it to a darker color (Morel and Machado 1981).
Average yield losses due to iron toxicity range from 35 to 45% (Lantin and Neue
1989; Audebert and Sahrawat 2000). Iron toxicity symptoms can appear in any
plant developmental stage. However, the end of tillering and beginning of flowering
are the stages in which the symptoms appear more frequently and clear (van
Mensroort et al. 1985; Fageria 1984). If iron toxicity occurs in the early stages of
development, plants suffer a severe retard in growth; when it is later, vegetative
growth is not much affected, but grain yield is reduced due to spikelet sterility
(Lantin and Neue 1988). However, some reports state that when iron toxicity occurs
in the beginning of the cycle, plant growth can be strongly affected and a total loss
of yield can occur (Abifarin 1988).
Analyzing the symptoms described by different authors, one can observe that there
is not a unique symptom characterizing iron toxicity, but a range of colors from
yellow to orange, with or without dark brown spots. In all descriptions, these symp-
toms start on older leaves and evolve from tip to base of leaf limb (Sousa et al. 2004).
Leaves become chlorotic because iron is needed for the synthesis of some
chlorophyll–protein complexes in the chloroplast. The low mobility of iron is due
to its precipitation in older leaves as insoluble oxides or phosphates or the formation
of complexes with phytoferritin, an iron-binding protein (Oh et al. 1996). Iron

precipitation decreases the metal’s subsequent mobilization inside the phloem. This
type of toxicity is less common in Brazilian conditions, but is frequently seen in
other climates, where some soils develop extremely high levels of Fe2+ when
flooded. Indirect toxicity results from the limited absorption of several nutrients
such as calcium, magnesium, potassium, phosphorous, and iron itself, due to iron
precipitation on rice root epidermis. The formation of an oxide–hydroxide Fe3+
layer on the root blocks nutrient absorption, resulting in multiple nutritional defi-
ciencies. Symptoms of this deficiency include plant atrophy, tillering reduction,
orange leaves, and the covering of roots by red layers of iron oxides. Besides iron
deposition in the roots, changes in leaf peroxidase activity have been described
(Peng et al. 1996; Fang and Kao 2000).
The insolubility of iron plus its high reactivity can cause severe damage to the
plant cell. The production of reactive species of oxygen, specifically the hydroxyl
radical (OH ), through the Fenton Reaction, is the major cause for its toxicity inside
the cell (Hell and Stephan 2003):
Fe3þ þ O2 ! Fe2þ þ O2
Fe2þ þ H2 O2 ! Fe3þ þ OH þ OH
Or:
O
2 þ H2 O2 ! O2 þ OH þ OH

The entrance of iron into the radicular symplast via the membrane transport
systems creates a need to once more protect it from oxygen. Protection is necessary
in order to avoid precipitation and the generation of reactive oxygen species.
Among the major chelating agents, nicotianamine (NA) appears as the best candi-
date because it forms poor Fenton reagent stable complexes with iron at both
oxidation states, its ubiquitous character, and its correlated localization with iron
(Stephan and Scholz 1990; Scholz et al. 1992; Stephan et al. 1996; Herbik et al.
1996; Liu et al. 1998; von Wiren et al. 1999; Pich et al. 2001).
After zinc, iron is the element that most frequently limits rice production, when
nutritional disorders in rice caused by micronutrients in Brazilian soils are assessed.
Two contrasting scenarios exist: one in dry conditions (upland rice) when the
problem is related to iron deficiency and the other in flooded conditions, due to
toxicity (irrigated rice). Increases in the Fe2+/Fe3+ ratio caused by reduction in the
flooded soil are the major cause of toxicity. This reduction can cause an increase of
6,000-fold in soluble iron (600 vs. 0.1 ppm) when soil redox potential reaches
100–300 mV (Brennan and Lindsay 1998).
In Brazilian soils commonly cultivated with flooded rice, soluble iron content
after flooding does not reach such high levels as registered in other traditional rice
growing countries. Generally, the iron content in Brazilian soils does not exceed
100 ppm. However, these levels are sufficient to cause iron toxicity in rice (Barbosa
Filho et al. 1994). The iron content in which toxicity occurs in the soil and plant
ranges between 10 and 1,000 ppm and 50 and 1,700 ppm, respectively. Such broad
limits illustrate that toxicity development is a complex phenomenon. It does not
appear that there is a specific factor in either the soil or the plant that allows a
prediction of toxicity (Barbosa Filho et al. 1994).
The predominant and therefore the most important form of toxicity in Brazil is
indirect. Toxicity due to the ferric form (Fe2+) can cause considerable losses in rice
production. This is specially the case in the acid soils of tropical and subtropical
areas (Fageria and Rabelo 1987; Wu et al. 1998), as found in southern Brazil. These
regions are characterized by their richness in iron and low pH (Silva et al. 2003).
Occurrence in rice fields may cause reductions in productivity as high as 80%
(Sousa et al. 2004). Iron toxicity was first detected in Brazil during the 1970s. The
introduction of modern-type rice cultivars, some of which showed sensitivity to
the excess of iron in the soil, revealed the problem. The problem was also seen in the
states of Santa Catarina, Minas Gerais, Rio de Janeiro, Espı́rito Santo, Goiás, and
Rio Grande do Sul (Sousa et al. 2004; Vieira et al. 1999).
7.2.2 Iron Metabolism
The stable forms of iron participating in plant metabolism are Fe2+ and Fe3+
(Staiger 2002). The oxidation of iron-carrying compounds is constantly detected,
iron going from Fe2+ to Fe3+ during the electron transfer and vice versa. The
complex compounds formed with iron such as Fe–S proteins are key to electron
transfer in the respiratory functions in mitochondria and in the photosynthesis
apparatus in the chloroplasts (Balk and Lobreaux 2005). Fe–S clusters also partici-
pate in nitrogen fixation, DNA repair, and metabolic pathways. Iron is an essential
component of different enzymes involved in electron transfer (redox reactions),
such as cytochromes, both heme and non-heme groups, as well as electron carriers
and ferredoxin, a substance known to be involved in the photosynthesis electron
transfer (Barbosa Filho 1994; Briat et al. 1995; Briat and Lobréaux 1997; Briat et al.
2007). The presence of iron was also observed in plant hormone synthesis as a
cofactor (Bouzayen et al. 1991; Siedow 1991). Iron is predominantly present in the
chloroplasts as phytoferritin and ferredoxin (ca. 75%) protein complexes which are
known to be involved in the photosynthesis electron transfer (Brown et al. 1972).
7.2.3 Iron Uptake
The predominant form of iron is the divalent form Fe2+. Its content in the soil ranges
from near zero up to 40% in the Fe2O3 form. In order to cope with the low solubility
of ferric ions, an active mechanism to release/absorb iron from Fe3+ oxide hydrates
to the soil solution is required. Due to their immobility, plants face a range of iron
availability in the environment. Both iron deficiency and toxicity are responsible
for severe nutritional disorders deeply affecting their physiology (Ponnamperuma
et al. 1955; Chaney et al. 1972). In general, two strategies, one based in reduction
and another based in chelation (Kim and Guerinot 2007), have been described for
the uptake of iron.
7.2.3.1 Strategy I (Reduction Based)
In this strategy, plants release protons into the surrounding rhyzosphere via a
proton-ATPase. Dicot plants improve iron absorption by three reactions: (1) proton
efflux via ATPase to acidify the medium and therefore increase Fe3+ solubility; (2)
reduction of Fe3+ by a Fe3+-reductase to a more soluble form Fe2+; (3) transport of
Fe2+ by an iron transporter (R€omheld and Marschner 1986).
7.2.3.2 Strategy II (Chelation Based)
The organisms using this strategy release phytosiderophores (PSs) that chelate Fe3+
at the rhizosphere, allowing specific protein transporters to import the Fe3+–PS
complexes (R€ omheld and Marschner 1986; Hell and Stephan 2003). Microorgan-
isms, as well as grasses, use this strategy. Yeast, although not secreting its own
siderophores, can recognize and absorb bacterial siderophores such as catecholate
or hydroxamate (Yun et al. 2000a; Yun et al. 2000b).
7.2.4 Iron Transport and Signaling
Iron uptake and transport have been described in the model eukaryote Saccharomy-
ces cerevisiae (Curie and Briat 2003). In the plasma membrane, reductases reduce
Fe3+ to Fe2+, which is more soluble. A flavocytochrome (Fre1p) reduces Fe3+ at the
cell surface. Many paralogs of the FRE gene have been found (FRE2 – FRE7) as a
result of yeast genome sequencing (Johnston et al. 1997). FRE2 encodes a protein
related to Fre1p while FRE3 and FRE4 genes are involved in the reduction of Fe3+-
siderophore (Dancis et al. 1990). When the cells are replete with iron, a low-affinity
uptake system is responsible for ferrous iron uptake. This is achieved by a plasma
membrane transport protein encoded by the FET4 gene (Dix et al. 1994; Dix et al.
1997). On the other hand, the genes FET3 and FTR1 play an important role in high-
affinity ferrous uptake, which is induced under iron-deficiency conditions (Askwith
et al. 1994; Stearman et al. 1996). FET3 encodes a trans-membrane protein from a
family of multicopper oxidases that has an oxidase catalytic domain located on the
cell surface. FTR1 encodes a plasma membrane permease containing a REGLE
motif that has been identified in the ferritin iron-storage protein and seems to be
responsible for an iron selective pore. A model for high-affinity iron uptake has
been proposed (Eide 1998). It requires that Fe2+ produced by the Fe3+ reductases be
oxidized outside the cell by the FET3p multicopper oxidase into Fe3+, which then
binds to a Fe3+-binding site on FTR1p. Then, a conformational change is caused by
this binding, enabling Fe3+ to be transported to the cytoplasm. On another model
species, rice, a survey on the iron homeostasis-related genes revealed 18 YS,
2 FRO, 13 ZIP, 8 Nramp, and 2 Ferritin genes (Gross et al. 2003).
The Nramp (Natural Resistance-Associated Macrophage Protein) family of metal
transporters is conserved from bacteria to mammals (Gunshin et al. 1997). How-
ever, these proteins have also been shown to transport Ni, Zn, Cu, Co, and Cd, as
well as Fe and Mn (Gunshin et al. 1997). In order to avoid imbalances in nutrient
supply and to meet the nutritional demands for the entire plant, vascular plants
employ a strategy of interorgan signaling (Schmidt 2003). The signal for systemic
regulation of root responses to iron has been suggested to be ITP1, an iron-binding
member of the LEA (late embryogenesis abundant) protein family (Krueger et al.
2002). Transcription factors induced by iron deficiency have been reported, includ-
ing 14-3-3 and zinc-finger proteins in barley (Negishi et al. 2002). Also, a protein
containing a helix-loop-helix domain, FER, was cloned from a tomato mutant (fer).
This mutant does not respond to iron deficiency and can only survive with a heavy
supply of iron chelates (Ling et al. 2002). Nitric oxide (NO) is responsible for the
translation of the Fe-deficiency signal, a ubiquitous signal in mammals and plants
(Wendehenne et al. 2001).
The transport of iron to the cell interior creates the necessity of a proper storage
in order to avoid possible damage due to reactive oxygen species. Iron is stored in
the apoplastic space, between the plasmatic membrane and the cell wall of plant
cells, in mitochondria (Zancani et al. 2004), in plastids (Seckback 1982), and in the
vacuole, in low pH and high organic acid concentrations (Briat and Lobréaux
1997). The vacuole is the place for iron and other metal sequestrations, either as
a mechanism of detoxifying the cell or as metal reservoir. Exactly how the vacuole
contributes to iron metabolism is not clear. Mutations that affect vacuolar function
also affect the assembly of high-affinity transport systems present in the plasma
membranes (Urbanowski and Piper 1999). Ferritin, a specialized iron-storage
protein, is used to store iron in both mitochondria and plastids. They consist of 24
subunit hollow spheres capable of storing up to 4,500 atoms of iron per molecule in
a soluble and bio-available form (Balla et al. 1992; Harrison and Arosio 1996;
Connolly and Guerinot 2002). Ferritin forms gated pores, which are highly con-
served in ferritins of humans down to bacteria. These pores control iron flow to
chelators (Liu and Theil 2005). Iron controls the transcription of plant ferritins
in soybean and maize (Fobis-Loisy et al. 1996; Wei and Theil 2000). Also, the
accumulation of plant ferritin is regulated post-transcriptionally, since ferritin
mRNA accumulates in the maize mutant ys1 to a similar level as in other genotypes
(Fobis-Loisy et al. 1996) but iron accumulation in leaves is lower.
Many genes involved in iron transport have been described. Two ZIP family
members that function as root iron transporters, IRT1 and IRT2, are responsible for
iron uptake from the soil in Arabidopsis (Eide et al. 1996; Guerinot 2000; Connolly
et al. 2002; Vert et al. 2002; Varoto et al. 2002). The OsIRT1 and OsIRT2 genes from
rice are predominantly expressed in roots and induced in low-Fe conditions

(Ishimaru et al. 2006). A root iron-chelate reductase, FRO2 (homologous to
FRE1, FRP1 and gp91phox), complements the Arabidopsis frd1 mutant, deficient
in root ferric-reductase activity (Robinson et al. 1999). Members of the Nramp
family, Nramp1, 3, and 4, are divalent metal transporters which tend to show
increased mRNA accumulation in Fe deficiency (Curie et al. 2000; Thomine et al.
2000). AtNramp3 is a vascular metal transporter involved in plant responses to iron
deficiency. It is expressed in the vascular bundles of roots, stems, and leaves under
Fe-sufficient conditions, suggesting a function in long-distance metal transport
within the plant (Thomine et al. 2003). Mobilization of vacuolar iron is essential
for seed germination on low iron and is performed by the products of genes
AtNramp3 and AtNramp4 (Lanquar et al. 2005). Some iron efflux transporters
belonging to the IREG/Ferroportin family have been reported (IREG 1-3) and
show sequence similarity to mammalian iron efflux transporters (McKie et al.
2000). YS1, a Fe3+-phytosiderophore transporter, was cloned in maize from the
ys1 (yellow-striped) mutant (Curie et al. 2001). It was reported as a membrane
protein that mediates iron uptake. YS1 is able to translocate iron that is NA or PS
bound, and its specificity to iron seems to be several fold higher than that to copper.
No evidence was found for YS1 to be active in zinc transport (Roberts et al. 2004).
Arabidopsis has eight homologues, YSL 1-8. AtYSL1 is an important nicotianamine
seed loading. This gene was expressed in the xylem parenchyma of leaves, where it
was upregulated in response to iron excess, as well as in pollen and in young siliqua
parts (Le Jean et al. 2005). AtYSL2 is a metal-regulated gene encoding a plasma
membrane transporter of nicotianamine–metal complexes that is expressed in many
cell types in leaves, roots, and reproductive organs showing a major role in the lateral
movement of metals in the vasculature (DiDonato et al. 2004). Rice has 18 putative
YS1-like genes exhibiting 36–76% sequence similarity to maize YS1. From these,
OsYSL2 is strongly induced in rice leaves by iron deficiency (Koike et al. 2004).
TcYSL3 is a Fe/Ni–NA influx transporter and a good candidate for the function of
entry of Ni–NA into the symplasmic transport in the root for delivering it into the
xylem. It is also important for the unloading of the Ni–NA complexes from the
xylem in the leaves and subsequent delivery to storage sites (Gendre et al. 2006).
A member of the LEA family, ITP, has a similarity to a Fe3+ polypeptide
chelating in the phloem (Krueger et al. 2002). A gene belonging to the cytb5
reductase family, an NFR homolog with iron reductase activity in the tonoplast
and in the phloem was reported (Bagnaresi et al. 2000; Xoconostle-Cazares et al.
2000). Four genes encode ferritin (AtFer1-AtFer4) in Arabidopsis. AtFer1 and
AtFer3 play important roles in the protection of plant cells from oxidative stress
(Petit et al. 2001). AtFer2 gene expression was detected in mature siliquas and dry
seeds, induced by ABA (Briat and Lobréaux 1997). Grasses that utilize strategy II
release a low molecular weight chelating compound such as mugineic acid (MA).
The phytosiderophore-Fe3+ complexes are then transported into the plant (Grotz
and Guerinot 2002). In this process, two genes are required for the conversion of
S-adenosyl methionine to Nicotianamine (Nicotianamine Synthase, NAS) and NA
to deoxymugineic acid (Nicotianamine Aminotransferase, NAAT). A shortage of
NA impairs the functions of metal-requiring proteins, including transcription fac-

tors (Takahashi et al. 2003). Maize has two types of NAS proteins based on their
expression pattern and subcellular localization (Mizuno et al. 2003). Three genes
were found: ZmNAS1, ZmNAS2, and ZmNAS3. The first two are expressed under
iron deficiency and the third is downregulated by iron deficiency and induced by
iron resupply. Three rice Nicotianamine Synthase genes, OsNAS1, OsNAS2, and
OsNAS3, have been shown to be expressed in cells involved in long-distance
transport of iron and differentially regulated by iron. OsNAS1 and OsNAS2 are
expressed in the vascular bundles of green leaves and in all cells showing chlorosis.
OsNAS3 expression is induced in roots but is suppressed in leaves in response to
iron deficiency (Inoue et al. 2003). A cDNA macroarray using 36 metal-related
genes from rice including metal transporter (ZIPs, NRAMPs, and YSLs) and metal
homeostasis (NAS, FER, FRO, NAAT, FDH, GSTU, and PDR) genes was developed
(Narayanan et al. 2007). The genes OsIRT1, OsZIP1, OsZIP5, OsZIP8, OsYSL5,
OsYSL6, OsYSL7 OsYSL8, OsYSL18, OsNramp2, OsNramp4, and OsNramp7 were
found to be expressed in all types of leaves (flag and non-flag).
7.2.5 Improving high/low Iron Tolerance in Rice
Rice is a particularly interesting species since it is described as a strategy II plant,

but it also absorbs iron through strategy I. This means that it can absorb iron via
chelated- and reduction-based strategies. The latter causes an acidification of the
medium and increases the ratio of soluble/insoluble iron in the soil (Ishimaru et al.
2006). Thus, rice has the advantage of plasticity regarding growing under normal or
submerged conditions. In general, plant species differ regarding the ability to
absorb nutrients, the degree of resistance to toxic elements, and efficiency in the
use of absorbed nutrients (Clark 1983; Furlani et al. 1986). Shoot length and 9 days
of stress were shown to be the best traits for discriminating Brazilian irrigated rice
genotypes (Crestani et al. 2009) regarding their genetic response to iron toxicity.
A mapping population consisting of 123 double-haploid (DH) lines was devel-
oped from a cross between IR64 and Azucena (Guiderdoni et al. 1992). The parents,
123 DH lines, and 100 DHBC1F1 (DH lines backcrossed to Azucena) were used to
find markers associated to seedling tolerance for ferrous iron toxicity (Wu et al.
1997). From a total of 175 cDNA and genomic clones tested, four marker loci on
chromosome 1 were identified to be significantly associated with both segregations
of tolerance index value (degree of bronzing) and RDSDW (relative decrease in
shoot dry weight). A significant association between one marker locus and RDSDW
was found. Also, QTLs explaining 32% and 15% of the tolerance index value and
15%, 21%, and 10% of the RDSDW were found (Wu et al. 1997). Another mapping
population consisting of 96 backcross inbred lines (BILs) derived from a cross
Nipponbare/Kasalath/Nipponbare was developed (Wan et al. 2003). The 96 BIL
lines in BC1F9 were phenotyped for iron tolerance. Four QTLs were detected using
RFLP markers on chromosomes 1 and 3 that were significantly associated with leaf
bronze index, stem dry weight, tiller number, and root dry weight.
Regarding iron defficiency, rice produces less phytosiderophores than wheat and
barley. One strategy has been to increase its PS production. When transgenic rice
plants expressing barley NA Aminotransferase were tested, their tolerance was
improved, achieving higher vigor and fourfold higher grain yield (Takahashi et al.
2001). The constitutive expression of two Fe3+--chelate reductases from yeast in
transgenic tobacco resulted in fourfold increase in iron reductase activity and 50%
increase in leaf iron content (Samuelsen et al. 1998). Constitutive expression of the
Arabidopsis NA Synthase gene resulted in a twofold to fourfold increase in leaf iron
content of tobacco plants, which grew faster and performed more efficiently under
iron-deficient conditions (Douchkov et al. 2001). However, improving iron uptake
alone is not sufficient, because of rate-limiting steps further in the pathway. On
the other hand, the increase of NA synthesis may be a viable option, although
co-supression has been observed in rice transformed with the barley NAS gene
(Mori et al. 2001).
7.2.6 Mutation Inducing
Another strategy to obtain improved genotypes for iron toxicity tolerance is mutation
inducing. Gamma ray was used to generate a collection of rice mutant genotypes
from the indica cultivar BR-7 “Taim” (Table 7.1). These mutants were screened for
many abiotic stresses, including iron, aluminum, organic acids, and root morphology
(Zimmer et al. 2003). Seven variables were analyzed on plants under iron stress:
number of roots (NR), main root length (MRL), coleoptile length (CL), shoot length
(SL), first leaf insertion (FLI), first leaf length (FLL), second leaf length (SLL).
Mutant 6 showed one of the best relative performances being constantly among the
three higher values in six of seven evaluated variables (NR, CL, FLI, FLL, SLL, and
APL). It also showed the highest values in four variables (FLI, FLL, SLL, and APL),
showing great potential as an iron-tolerant genotype. Mutants 4 and 7 were also
promising, as both were in the top three values of relative performance in four of
seven evaluated characters (FLI, FLL, SLL, and APL; CL, FLI, FLL, and APL,
respectively). Mutant 26 was among the three higher values of relative performance
in three of seven evaluated characters (NR, MRL, and CL). These mutants show
promise for studying iron uptake and metabolism and are being further investigated.
7.3 Toxicity of Organic Acids to Irrigated Rice
7.3.1 Organic Acid Genesis in Flooded Soils
Soil flooding decreases gas exchanges between air and soil, since the diffusion of
gases in water is ca. 10,000-fold lower than that in the air. As a consequence,
oxygen supply to the soil is very slow and below microorganism needs. In this
condition, facultative and obligatory anaerobic bacterial microorganisms prolifer-
ate and dominate the biological activity (Ponnamperuma 1972; Sousa et al. 2004).
In the absence of oxygen, biochemical processes responsible for the organic acid
metabolism in flooded soils are anaerobic respiration and fermentation. In the
anaerobic respiration, microorganisms use the energy released from organic carbon
oxidation in their vital processes and from inorganic compounds (nitrate, oxides
and manganese hydroxide, iron, and sulphate) such as electron receptors.
In fermentation, media organic compounds or byproducts of metabolic routes
are used as donors and acceptors of electrons in the oxirreduction process. These
organisms do not use an electron transport chain to oxidate NADH to NAD+,
but should work through an alternative form to use this energy and maintain
a supply of NAD+. Fermentation is characterized by a smaller generation of CO2
and the formation of short chain and low molecular weight organic compounds.
Despite being an inefficient way of breakdown, fermentation promotes the break of
complex organic substrates, resulting in a series of substances, many of them
transitory and not found in oxidized soils. Many of these substances have the
potential of causing toxicity to irrigated rice, especially the short-chain organic
acids, such as acetic, propionic, and butyric acids (Rao and Mikkelsen 1977).
The anaerobic decomposition of organic compounds happens in successive
steps involving different groups of microorganisms which convert complex mole-
cules in simpler forms, those described in Fig. 7.3 (Silva et al. 2008). In the
beginning of the process, there is a hydrolysis of organic polymers of plant origin
(plant tissue components) into monomers (such as carbohydrates into glycids,
lipids into long-chain organic acids, and proteins into aminoacids). This occurs
because facultative or obligatory anaerobic microorganisms secrete extracellular
enzymes, transforming complex compounds into simpler ones. These simple chain
organic compounds are assimilated by these microbes and fermented intracellu-
larly into short-chain organic acids, such as the acetic (CH3COOH), propionic
(CH3CH2COOH), and butyric (CH3CH2CH2COOH) acids, in a process called acid
formation. Following this process, there is the production of acetic acid, from
organic acids with more than two carbons. This step is called ketogenesis and is
regulated by anaerobic microorganisms that cannot convert acetic acid into CH4
due to enzymatic limitations. At the end, CH4 is formed from simple compounds
generated by ketogenesis as well as formate, H2, methanol, methyl amines, and CO2.
The production of organic acids in flooded soils is directly proportional to
degradable carbon availability. Thus, soils rich in organic matter or those soils in
which organic residues are added close to the flooding condition tend to present
higher production of organic acids. The organic acids can start to accumulate in
flooded soils where organic residues have been deposited as soon as day one.
Commonly, acid concentration is low at the first few days, reaching maximal values
between 2 and 4 weeks of flooding (Sousa et al. 2002). Then, the acid concentra-
tions decrease until stable and low values are found (Fig. 7.4). The peak of acid
release varies as a function of soil characteristics, residue amounts, and the type of
acid evaluated.
Organic Macromolecules
Hidrolysis
Despolimerization
Proteins Carbohydrates Lipids
Aminoacids Glicids Long chain Organic acids
Acidogeneis
Short chain Organic acids
Butyric acid Propionic acid Acetic acid Formic acid
CO2 and H2 CH4
Fig. 7.3 Scheme showing the degradation of organic matter into simpler compounds in flooded
soils. Adapted from Silva et al. (2008)
7.3.2 Organic Acid Toxicity Symptoms
The toxicity by organic acids in rice is observed at early stages of plant develop-
ment, characterized by a lower germination percentage, lower radicle development,
and lower plant height and weight (Sousa and Bortolon 2002). In cases of severe
toxicity, plant growth injuries can reflect in other phases, leading to decreases in
tillering ability, nutrient absorption, and grain yield (Camargo et al. 1993; Camargo
et al. 2001). The higher toxic effect of organic acids occurs in the root system, and
concentrations of 2.5 mmol L1 acetic, 1.25 mmol L1 propionic, and 1.00 mmol
L1 butyric acid are capable of causing significant reductions on rice growth (Sousa
and Bortolon 2002; Schmidt et al. 2007), as can be observed in Fig. 7.5.
The monocarboxylic acids (such as acetic, propionic, and butyric) alter the
composition of organic acids on the plasma membrane, decreasing the ratio of
2000 2 cm
5 cm
10 cm
1500
Acetic acid, mg L–1
1000
500
0
0 7 14 21 28 35 42 49 56
Days of flooding
Fig. 7.4 Acetic acid contents in the soil solution at three depth measures in a flooded Albaqualf,
with ammending of ryegrass residues in amounts equivalent to 10 Mg ha1. Adapted from Sousa
et al. (2002)
polyunsaturated acids, affecting an important property of the membrane such as

selectivity and increasing solute leaking (Marschner 1995). Therefore, organic
acids can harm the development of the crop, mainly by inhibiting root elongation
and nutrient absorption (Takenaga 1995; Sousa and Bortolon 2002). Organic acids
cause root cell division inhibition at the point of contact between root and acid
(Armstrong and Armstrong 2001).
Critical levels of organic acid toxicity reported in the literature vary as a function
of time of exposure of plants to organic acids, nutrient concentration and nutritive
solution pH (Fortes et al. 2008), genotype (Kopp et al. 2008), and acid used, making
the establishment of a standard toxic concentration difficult. A concentration of
4.7 mmol L1 of acetic acid causes 50% reductions on root growth of rice cultivar
BRS-7 “Taim”(Sousa and Bortolon 2002). A similar result was observed with
1.7 mmol L1 propionic and 2.0 mmol L1 butyric acid (Schmidt et al. 2007).
On the other hand, Kopp et al. (2007a) found that concentrations of 10.9 mmol L1,
5.6 mmol L1, and 5.3 mmol L1 of acetic, propionic, and butyric, respectively,
were needed to achieve the same 50% reduction of root growth for cultivars BRS-7
“Taim” and SAIBAN.
Even with some differences among authors about the critical levels of organic
acid toxicity in irrigated rice, there is a common view that acetic acid, although
present in higher amounts in flooded soils, shows less toxicity than propionic and
butyric. The increase in the number of carbons on the chain increases the degree of
toxicity of the organic acid (Takijima 1964; Rao and Mikkelsen 1977). However,
such difference is not so clear when one compares propionic and butyric acid
(Schmidt et al. 2007; Kopp et al. 2007a).
Fig. 7.5 Rice plants subjected to different organic acid concentrations in nutrient solution for
13 days.
Studies developed in our group regarding organic acid tolerance in rice cultivars
and mutants demonstrate that many distinct mechanisms do exist for tolerance to
each of the major acids formed in the soil (acetic, propionic, and butyric). It was
shown that genotypes tolerant to one acid do not necessarily tolerate the other two
(data not shown). However, some genotypes show tolerance to more than one acid
or even to the three of them. When these results are compared to studies where
all three acids were added simultaneously to form the treatments (Wallace &
Whitehand 1980), one observes that the proportion of tolerant genotypes is reduced.
In order to study the genetic variability for tolerance to organic acids in rice, a
mutant population was screened (Zimmer et al. 2003; Kopp et al. 2007b, c, d). After
cycles of generation advancing, 40 lines were obtained for genetic studies. Lines
were divided in 25% tolerant to acetic and propionic and 27.5% tolerant to butyric
acid. Also, some very sensitive lines were identified. These results suggest that the
mutagen affect some genes related to organic acid response. In Oat, mutants
obtained from a gamma Ray induction in the oat cultivar UFRGS 14, which is
sensitive to organic acids (Kopp et al. 2006), were shown to vary regarding
tolerance to these compounds. The evaluation of 30 mutant lines resulted in
23.3% tolerant genotypes. Further studies regarding mapping and inheritance of
these genes are under way.
7.4 Conclusion and Perspectives
The genomic analysis of plant roots will enable us to better understand abiotic
stresses and improve iron tolerance and/or accumulation as well as organic acid
tolerance. Rice is the major staple food for over half of the world’s population and
understanding the major stresses affecting the rice crop will enable scientists to
design better plants with better yields in order to feed the growing population and
save the occupation of virgin areas today maintained as ecological reserves. Deal-
ing with iron and organic acids is not a simple task, and a better understanding of
the mechanisms by which plants absorb, transport, and store/process these com-
pounds will allow better land use and management. Root genomics is likely to be
among the major sciences in this century, since roots have been largely neglected
despite its importance on the plant vs. environment interactions.
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Chapter 8
Genomics of Root Architecture and Functions
in Maize
Roberto Tuberosa, Silvio Salvi, Silvia Giuliani, Maria Corinna Sanguineti,

Elisabetta Frascaroli, Sergio Conti, and Pierangelo Landi
Contents
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
8.2 QTLs for Root Architecture and Associated Traits in Maize . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
8.2.1 Effects of the QTL Region on Bin 2.04 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
8.2.2 Effects of the QTL Region on Bin 1.06 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
8.2.3 QTLs for Root Architecture of Maize Grown Under Environmentally
Constrained Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
8.3 Production and Characterization of Near Isogenic Lines for QTLs for Root Traits . . . . 188
8.3.1 Effects of Root-ABA1 on Root Architecture, ABA Concentration,
Root Lodging, and Grain Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
8.3.2 Identifying Candidate Genes for Root Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
8.4 “Omics” of Maize Root Development and Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
8.5 Conclusions and Challenges Ahead . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
8.1 Introduction
Twenty-first century agriculture will face formidable challenges to provide man-

kind with an appropriate level of food security while enhancing the sustainability
and profitability of agricultural practices, lowering their environmental impact,
and preserving the remaining biodiversity (Borlaug and Dowswell 2005). These
challenges will be even more daunting in view of the increased unpredictability of
weather patterns as a result of global climate change and the decreased availability
of irrigation water required for mitigating the negative effects of drought (Pennisi
2008).
Among the major crops that feed mankind, maize is expected to become the most
important by 2030, especially in view of the projected increase in the demand for feed
in meat production. More severe and frequent droughts have been forecasted
R. Tuberosa (*), S. Salvi, S. Giuliani, M.C. Sanguineti, E. Frascaroli, S. Conti, and P. Landi
Department of Agroenvironmental Sciences and Technology, Viale Fanin 44, 40127 Bologna, Italy
e-mail: roberto.tuberosa@unibo.it

180 R. Tuberosa et al.
for regions where maize represents an important component in the human diet (e.g.,
tropical Africa and Northeast China) or for biofuel and livestock production
(e.g., USA and Eastern Europe). In this challenging scenario, better knowledge of
the genetic and functional basis of the processes regulating the development and
plasticity of maize roots will allow for a more effective selection to improve yield
potential while optimizing water- and nutrient-use efficiency (Guo et al. 2005b; Bohn
et al. 2006; de Dorlodot et al. 2007; Osmont et al. 2007; Yu et al. 2007; Desnos 2008;
Hochholdinger and Tuberosa 2009). Root traits have been shown to play a major role
in the adaptive response of crops to drought and low nutrients (Tuberosa et al. 2003;
Lynch 2007), and their selection has often been advocated to mitigate yield losses in
crops exposed to water and nutrient deficits (Ludlow and Muchow 1990). This
notwithstanding, breeders have largely neglected selecting for roots, not only for the
demanding phenotyping but also for the difficulty in identifying a yield-effective
ideotype and to effectively select the desirable root architectural features. Other
factors that have traditionally discouraged root studies in field-grown plants are the
low heritability of root features consequent to high soil heterogeneity and the need to
utilize destructive approaches. Maize is no exception to the above.
As an alternative to root surveys in field-grown plants (Fincher et al. 1985; Beck
et al. 1987), studies implemented under controlled conditions (e.g., hydroponics,
aeroponics, pots) at an early stage facilitate the measurement of root characteristics
in a large number of plants (Nass and Zuber 1971; Arihara and Crosbie 1982; Stamp
and Kiel 1992; Landi et al. 1998; Sanguineti et al. 1998, 2006). Nonetheless, the
unnatural environment in which roots grow and the early growth stage that is
usually considered in such studies are major shortcomings that should be cautiously
considered before extrapolating the results to field-grown plants. In maize, a
significant, albeit weak, positive association was reported between seminal root
traits in hydroponics and root-pulling resistance in the field (Landi et al. 2001).
Additionally, seminal roots in maize play a prominent role in nutrient acquisition at
the seedling stage and thus influence early vigor, a feature particularly relevant
under conditions of zero or minimum tillage characterized by low agronomic input.
The length and number of seminal roots may be particularly important in the
acquisition of immobile nutrients such as phosphorus (Kaeppler et al. 2000; Zhu
et al. 2005a, b, c, 2006; Lynch 2007). As the plant reaches flowering, the importance
of seminal roots declines as compared to shoot-borne roots, commonly
named adventitious nodal roots (Kiesselbach 1949; Hochholdinger et al. 2004b),
which have been shown to positively affect grain yield in water-limited conditions
(Duchoslav et al. 1989; Navara et al. 1993, 1994; Jesko 2001).
Maize roots show a high level of developmental plasticity in response to external
cues (Hose et al. 2000; Ito et al. 2006), a clear example being provided by the
interplay between abscisic acid (ABA) and ethylene in sustaining root elongation
under conditions of water deficit which inhibit shoot elongation (Sharp and Davies
1985; Saab et al. 1990; Zhang and Davies 1990; Sharp 2002; Sharp et al. 2004;
Spollen et al. 2008). Additionally, this plasticity insures the optimization between
the allocation of photosynthates to the root and its capacity to (1) capture water and
nutrients as a function of the prevailing soil conditions and (2) mitigate the negative
8 Genomics of Root Architecture and Functions in Maize 181
effects of adverse soil conditions. A clear example of the latter is provided by the
development of aerenchyma in adventitious roots in response to water-logging
conditions (Mano et al. 2005a, b).
Notwithstanding the important role of roots for optimizing maize yield (Bolaños
et al. 1993; Hammer et al. 2009), the genetic factors that control root growth have
only recently started to be unveiled with the use of mutants and, in some cases, their
cloning (Taramino et al. 2007; Hochholdinger et al. 2008). As an example, the
cloning of rtcs (rootless concerning crown and seminal roots) revealed its role in
encoding an auxin-inducible transcription factor that controls the early events
leading to the initiation and maintenance of seminal and shoot-borne root primordia
(Taramino et al. 2007). Nonetheless, because the genetic basis of the variability of
root architecture in cultivated maize is prevalently quantitative, the application of
suitable genomics approaches is required to identify the relevant quantitative trait
loci (QTLs). This, in turn, would enable breeders to apply marker-assisted selection
(Varshney and Tuberosa 2007) for tailoring roots according to the ideotype
perceived as optimal to maximize crop performance in the target environment
(de Dorlodot et al. 2007; Tuberosa et al. 2007).
In this context, the present review surveys the main findings of QTL studies
and other genomics approaches aimed at (1) dissecting the genetic basis of the
variability in root architecture in maize and (2) investigating and interpreting the
effects of this variability on yield and other agronomic traits.
8.2 QTLs for Root Architecture and Associated

Traits in Maize
The maize root system includes embryonic primary and seminal roots and postem-
bryonic shoot-borne and lateral roots (Hochholdinger et al. 2004b) which have
different functions as development progresses. As an example, at flowering, shoot-
borne nodal roots play a predominant role in extracting moisture from the more
superficial portion of the soil horizon, while primary and seminal roots allow plants
to access moisture more deeply stored and useful to avoid desiccation under drought
conditions. However, a large root system does not guarantee a high yield as shown in
the recurrent selection work in maize carried out at CIMMYT to improve grain yield
under severe drought conditions (Bolaños et al. 1993) and by a study conducted
testing families derived from the cross of inbred lines (B73 and Mo17) which
differed in root characteristics at an early growth stage (Bruce et al. 2002).
A comparative analysis of the results of QTL studies in maize is facilitated by
the availability of the UMC (University of Missouri, Columbia) reference map
which has been subdivided into 103 sectors (bins) of comparable size (Davis et al.
1999). The boundaries of each bin are defined by flanking markers (RFLPs and
SSRs) included in a public set of Core Markers (Gardiner et al. 1993; Davis et al.
1999). The UMC map reports over 15,000 loci and includes genes, probed sites,
cytological breakpoints, and QTLs (Schaeffer et al. 2006). Because the bin
framework integrates over 130 independent map sets and includes all mapped loci
stored in MaizeGDB (http://www.maizegdb.org), it has been used extensively for
the comparison of QTL positions across genetic backgrounds (Lin et al. 1995;
Khavkin and Coe 1997; Tuberosa et al. 2002b, 2003, 2005; Chardon et al. 2004;
Sawkins et al. 2004; Schaeffer et al. 2006; Wang et al. 2006). Gramene (http://
www.gramene.org) is another database that reports information on maize QTLs and
allows for comparative searches of maize genomics data with other grasses (Ware
et al. 2002). Importantly, the UMC map allows us to compare the map position of
mutants (Neuffer et al. 1997) with that of QTLs, thus contributing relevant infor-
mation for validating Robertson’s hypothesis for a specific locus (Robertson 1985).
The first comparative analysis of root QTLs in maize (Tuberosa et al. 2003)
highlighted the role of two major QTL regions (on bins 2.04 and 1.06) for their
effects on root architecture and other traits, including grain yield, in different
genetic backgrounds. In order to more accurately evaluate the effects of these two
QTLs on root traits and grain yield, near isogenic lines (NILs) differing for the
parental segment at these QTL regions have been developed (for bin 2.04 see Landi
et al. 2005; for bin 1.06: Landi et al. 2010). The main results reported to date for
these QTL regions are summarized hereafter while the results obtained with the
NILs for bin 2.04 are reported in Sect. 8.3.1.
8.2.1 Effects of the QTL Region on Bin 2.04
Lebreton et al. (1995) were the first to report the significant effect of bin 2.04 on
root architecture using an F2 population (81 plants in total) derived from the cross
between Polj17 and F-2, two lines that were known to differ for root features,
especially root-pulling force (RPF) at flowering, and also for the concentration of
ABA in the leaf and xylem sap. For all but one of the detected QTLs, the additive
effects for ABA concentration and RPF were concurrent. A remarkable correlation
(r ¼ 0.84) was found between the QTL effects for nodal root number and ABA
concentration in the xylem sap. The QTL region on bin 2.04 also showed the
strongest effect on leaf ABA concentration (L-ABA); this finding was confirmed
by Tuberosa et al. (1998) using a mapping population derived from the cross
between Os420 and IABO78, two lines widely different for drought tolerance and
L-ABA (Tuberosa et al. 1994; Landi et al. 2001). It is worth noting that none of the
major mutants impaired in ABA biosynthesis mapped in bin 2.04, a result that led
Tuberosa et al. (1998) to postulate that the effect of the QTL on L-ABA might have
been due to a primary effect on root size/architecture, hence on the water status of
the plant, the major factor influencing the concentration of ABA in plant tissues
(Quarrie 1991). Subsequent studies conducted to further characterize the effects of
this QTL in the Os420 IABO78 background have shown its marked influence on
root architecture, root lodging, and grain yield but not on the water status of the
plant (Giuliani et al. 2005b; Landi et al. 2007). Further details on the characteriza-
tion of the bin 2.04 QTL are provided in Sect. 8.3.1. Additionally, the meta-analysis
conducted by Sawkins et al. (2004) has highlighted the effects of bin 2.04 on grain
yield under conditions of water stress. Recently, the importance of bin 2.04 in
controlling root features has been reported by Trachsel et al. (2009) in an RIL
population derived from the cross between CML444 (drought tolerant) and SC-Ma-
lawi (drought sensitive) and tested for length of axile and lateral roots at 2, 5, 7, and
9 days after germination. In particular, a QTL region on bin 2.04 affected the
elongation rate of lateral roots as well as the elongation and number of axile roots.
Additional bins that affected root growth were also reported in bins 1.03, 1.04, 1.08,
2.05, and 7.04. Based on a comparative analysis of their results with those previ-
ously published, Trachsel et al. (2009) suggested that root growth at the juvenile
stage can be predictive of root morphology at later developmental stages.
8.2.2 Effects of the QTL Region on Bin 1.06
In an experiment conducted in hydroponics using 171 F3 families derived from the

cross Lo964 Lo1016, several QTLs were shown to influence primary root length
(R1L), primary root diameter (R1D), primary root weight (R1W), and the weight of
the adventitious seminal roots (R2W) (Tuberosa et al. 2002c). Bin 1.06 was the
chromosome region with the most sizeable QTL effects (LOD values of 14.7, 6.4,
and 8.3 for R1D, R1L, and R2W, respectively). In order to investigate to what
extent the QTLs influencing root growth in hydroponics may also regulate root
growth in the field, a random sample of 118 (Lo964 Lo1016) F3 families were
tested for root-pulling force (RPF) at flowering in replicated field trials (Landi et al.
2002). Out of the 30 bins with QTLs for RPF and/or number of brace roots,
15 (including bin 1.06) also harbored QTLs for root traits in hydroponics, i.e., a
frequency much higher than what would be expected based solely on chance.
Subsequent field trials conducted during two growing seasons to measure grain
yield (GY) under well-watered (GY-WW) and water-stressed (GY-WS) conditions
with the Lo964 Lo1016 F3 families revealed several QTLs whose peaks over-
lapped with those for root traits measured in hydroponics (Tuberosa et al. 2002c)
and/or in the field (Landi et al. 2002). In particular, QTLs for R2W co-localized
with QTLs for GY-WW and/or GY-WS in bins 1.03, 1.06, 1.08, 7.02, 10.04, and
10.07. At five of these six chromosome regions, an increased root weight was
associated with a higher GY, a result more likely to be due to pleiotropy rather
than linkage, in view of the number of independent chromosome regions involved
and the consistency of their effects. Of all regions which concomitantly influenced
root traits and GY, the strongest and most consistent effects were confined to a
10 cM interval on bin 1.06 that affected root features in both hydroponics and field
conditions and GY under both WW and WS conditions. QTLs for root traits on bin
1.06 have also been reported in Polj17 F-2 (Lebreton et al. 1995), B73 Mo17
(Kaeppler et al. 2000), F288 F271 (Barriere et al. 2001), and Z3 87-1 (Liu
et al. 2008a). Additionally, it is worth noting that Hirel et al. (2001) reported a
major QTL for nitrogen-use efficiency and GY on bin 1.06, a finding which further
highlights the importance of this region for GY.
8.2.3 QTLs for Root Architecture of Maize Grown Under

Environmentally Constrained Conditions
Because drought is the major environmental factor curtailing maize yield (Duvick
2005), a number of reviews have already surveyed QTLs for roots in maize under
water-limited conditions and their role in sustaining yield (Tuberosa et al. 2002b, c,
2003, 2007). Here, we summarize the main findings of the studies that have
investigated QTLs for roots of maize grown under low temperature, low nutrients,
flooding, or in the presence of root worms or in conditions that favor root lodging.
8.2.3.1 Root QTLs at Low Temperature
The development of a vigorous, highly structured root system might be of major

importance for growth at low temperature (Hund et al. 2008), especially in no-
tillage systems, where low soil temperature becomes a major limiting factor.
Genotypic differences in cold tolerance exist for the development of the root
(Stamp 1984). QTLs controlling root tolerance to cold at early stages were studied
in a set of 168 F2:4 families of the Lo964 Lo1016 cross derived from a
corresponding set of F2:3 families originally tested for root traits and tolerance to
drought (Landi et al. 2002; Tuberosa et al. 2002b). Seedlings were grown at
15/13 C and evaluated for shoot and root traits (Hund et al. 2004). The analysis
of root weight, length, and diameter led to the identification of 38 QTLs, seven of
which confirmed QTLs reported by Tuberosa et al. (2002b) for root traits in the
same population evaluated in hydroponics at normal temperature. A locus on bin
5.07 for root growth at low temperature was also shown to influence cold tolerance
at germination on the same mapping population (Frascaroli et al. unpublished
results), thus suggesting that this QTL region plays an important role in controlling
cold tolerance at different growth stages.
8.2.3.2 Root QTLs Under Low Nitrogen Conditions
The work of Wiesler and Horst (1994) demonstrated that a deeper root system is
essential in maize for utilizing nitrate in deep soils under field conditions and
showed that N-efficient maize cultivars had longer roots and larger root surface
areas.
An important aspect of maize productivity relates to the capacity of the plant to
efficiently absorb soil nitrogen, store it in the vegetative organs, and relocate it
during kernel growth (Wang et al. 2004; Chun et al. 2005; Hirel et al. 2007; Coque
et al. 2008). Although QTLs for nitrogen-use efficiency have been described and in
some cases accurately characterized in terms of biochemical effects (Agrama et al.
1999; Hirel et al. 2001, 2007; Gallais and Hirel 2004), their possible effects on root
architecture and functions remain to be duly investigated. Conversely, QTLs have
been identified for root hair length and plasticity in response to low phosphorus, a
nutrient that unlike nitrogen shows low mobility in the soil (Chassot and Richner
2002; Zhu and Lynch 2004). By enhancing soil exploration, root hairs play an
important role in the uptake of phosphorus.
A paper-roll culture system was used to investigate root hair length (RHL),
taproot length, root thickness, and root biomass in a RIL population derived from
B73 Mo17 (Zhu et al. 2005a, b). One QTL was associated with RHL plasticity,
three QTLs with RHL under high fertility, and one QTL with RHL under low
phosphorus. Six QTLs accounted for 53% of the total variation for seed phosphorus
content among RILs. Root biomass plasticity was significantly correlated with RHL
induced by low phosphorus, taproot length plasticity, and seed phosphorus reserves.
The only study that has extensively investigated root QTLs under different
nitrogen levels was conducted by Liu et al. (2008a) using 94 RILs derived from
the cross Z3 87-1, a hybrid widely grown in China. The lateral root length
(LRL), axial root length (ARL), maximal axial root length (MARL), axial root
number (ARN), and average axial root length (AARL) were evaluated under low N
(LN) and high N (HN) conditions in a hydroponics system. Of the 17 QTLs that
were detected by Liu et al. (2008a), 14 were located on chromosome regions where
other authors had previously reported QTLs for root architectural features (Lebreton
et al. 1995; Guingo et al. 1998; Landi et al. 2002; Tuberosa et al. 2002b; Hund et al.
2004; Mano et al. 2005a, b; Zhu et al. 2005a, b, 2006). Unexpectedly, among these
17 QTLs, no common loci were found under both LN and HN conditions for any
root traits, one possible reason being that the RIL population for QTL detection in
this study was very small. A major QTL on bin 1.06 (between bnlg1025 and
umc2029) for the AARL under LN explained 44% of the phenotypic variation
and co-localized with previously described QTLs for grain yield under low nitrogen
(Agrama et al. 1999; Bertin and Gallais 2001) and water-limited (Tuberosa et al.
2002c) conditions as well as for a number of root architectural features (Tuberosa
et al. 2002b; Zhu et al. 2006; Landi et al. 2010). Other striking coincidences were
identified on (i) chr. 8 between a QTL for LRL at HN (umc1997/umc1724) and the
QTL for LRL at high phosphorus supply (tpi5/umc07) reported by Zhu et al. (2005b),
and (ii) chr. 10, between a QTL for ARN (umc2043/umc1061) and a QTL for seminal
axial root number (pgamctg300/umc49b/umc44a) reported by Hund et al. (2004).
8.2.3.3 Root QTLs Under Low Phosphorus Conditions
Phosphorus (P) deficiency of soils can be a major yield-limiting factor in maize

production, particularly in low-input agriculture and in developing countries.
In maize, QTL studies have shown the importance of length and number of lateral
and seminal roots in the acquisition of phosphorus (Kaeppler et al. 2000; Zhu et al.
2005a, b, c, 2006; Lynch 2007; Hao et al. 2008). At low soil P concentration, plant
growth is affected both by physiological factors inherent to the crop as well as by
their interactions with the soil biota. Among the different species colonizing the
soil, the role of mycorrhiza in nutrient uptake of crops remains largely unknown.
Kaeppler et al. (2000) identified QTLs for growth at low P and response to
mycorrhizal fungi in a (B73 Mo17) RIL population. Three QTLs influenced
growth and shoot weight at low P in the absence of mycorrhizae and one QTL
on chr. 2 controlled mycorrhizal responsiveness. QTLs for root volume detected
for the high-P treatment were not coincident with any of the QTLs detected at
low-P concentration.
Following a study of root hair length in hydroponics under low P, Zhu et al.
(2005a, b, 2006) identified a major QTL flanked by npi409-nc007 on chr. 5. Chen
et al. (2008) evaluated 241 (Ye107 082) F2:3 families under normal phosphorus
(50 kg P/ha) and low phosphorus (0 kg P/ha) conditions at two sites. A total of 30
and 45 distinct QTLs were shown to influence growth and P efficiency in the
two sites. Three regions were found to influence relative root dry weight on bins
5.05 (mmc0282-phi333597 interval), 5.06 (umc1680-P5M1/c interval), and 5.07
(bnlg1346-bnlg1695 interval) at both sites. Each one of these QTLs explained
13–16% of the variation of relative root dry weight.
Another trait that has been suggested to influence P efficiency is root exudates
(Hinsinger 2001; Jones and Hinsinger 2008). Root exudates such as acid phospha-
tases, organic acid, and H+ compounds may help the mobilization of P from soils.
QTLs for P uptake in bean were found to influence H+ and total acid exudation from
the root (Yan et al. 2004), two processes capable of mobilizing soil-bound P
through soil P desorption or mineralization.
8.2.3.4 Root QTLs Under Flooding Conditions
Root features also play an important role in tolerance to soil flooding or water
logging (Ray et al. 1999; Mano et al. 2006a, b). The devastating flood of 1993 that
hit most of the corn-producing area in the Midwest USA caused $20 billion damage,
curtailing corn production by almost 30% and significantly raising the cost of corn-
based goods. Clearly, the availability of hybrids more tolerant to the negative effects
of soil anoxia caused by flooding would be beneficial for stabilizing corn production
and farmers’ income under such adverse conditions. A number of QTL studies have
investigated root features in response to flooding and water-logging conditions
(Mano et al. 2005a, b; Qiu et al. 2007). One of the major adaptations to soil flooding
is the adventitious root formation (ARF) at the soil surface. QTLs for ARF were
identified by Mano et al. (2005b) under flooding conditions in 110 F2 plants
derived from a cross between the dent line B64 with the tropical Caribbean
Flint line Na4. The QTLs for ARF were located on bins 3.07, 3.08, 7.04, 7.05,
and 8.05. At all QTLs, the Na4 alleles increased ARF. The comparison of ARF
QTLs in the B64 Na4 population with those in a B64 teosinte (Zea mays ssp.
huehuetenangensis) population showed the consistency of the QTLs on chr. 8 (Mano

et al. 2005a). Zea mays ssp. huehuetenangensis contributed all of the favorable QTL
alleles for ARF, thus supporting the conclusions of Campos et al. (2004) concerning
the value of mining genetic variation from outside cultivated maize to improve its
root architecture and functions. On a similar line, QTLs for aerenchyma formation
in roots, another important feature for adaptation to water logging, were identified
by using an F2 population generated from the B64 teosinte (Zea mays ssp.
nicaraguensis) cross (Mano et al. 2007; Mano and Omori 2008). Seedlings of
Zea mays ssp. Nicaraguensis clearly formed aerenchyma in the cortex of adventi-
tious roots in non-flooding conditions, whereas the maize inbred line B64 did not.
Four QTLs for aerenchyma formation under non-flooding conditions were located
on chr. 1 (Qaer1.02-3 and Qaer1.07), chr. 5 (Qaer5.09), and chr. 8 (Qaer8.06-7);
collectively, these regions accounted for 47% of the total phenotypic variance for
aerenchyma formation (Mano et al. 2007). Additional QTLs for root aerenchyma
under drained conditions have been described in a B73 teosinte (Zea luxurians)
population (Mano et al. 2008). Markers linked to QTLs for aerenchyma formation
in drained soil conditions could be used to develop maize hybrids with increased
flooding tolerance and greater yield stability under such conditions. Additionally,
increasing aerenchyma formation might improve soil exploration for a given
amount of dry matter invested in the root and might lower the metabolic cost for
maintaining root functions. Root and shoot traits were investigated in two experi-
ments to identify QTLs associated with water logging tolerance in an RIL popula-
tion derived from the cross HZ32 K12 (Qiu et al. 2007). Several QTLs for shoot
dry weigh, root dry weight, total dry weight, plant height, and water logging
tolerance mapped on chrs. 4 and 9. These QTLs were consistently detected in
both experiments. Secondary and more trait- or environment-specific QTLs influ-
encing water logging tolerance were also identified on chrs. 1, 2, 3, 6, 7, and 10.
8.2.3.5 Root QTLs Under Lodging Conditions
The evaluation of the historical series of maize hybrids released during the past 60
years indicates that modern hybrids are considerably more resistant to lodging than
older hybrids, particularly at high planting density, a condition that clearly accent-
uates this difference (Duvick 2005; Hammer et al. 2009). Lodging resistance is the
result of two components: one acting at the level of the root and one at the level of
the stalk. Mechanically, root lodging can be caused by strong wind, particularly
following heavy rains and/or by a weakened root system following the attack
of root worms. It has been shown that root architecture is a major factor influencing
root lodging (Ennos et al. 1993). Although a rather large genotypic variability
in root lodging has been reported in maize (Melchinger et al. 1986; Stamp and
Kiel 1992), the low heritability and unpredictability of root lodging in the field
coupled with the high cost required to carry out a large-scale evaluation using
artificial devices (Guingo and Hebert 1997) have traditionally hindered the
improvement of root lodging. Guingo et al. (1998) measured a number of root traits
for two seasons in 100 field-grown RILs from the cross between F-2 (root-lodging
susceptible) and Io (root-lodging resistant). The only QTL that concomitantly
influenced a number of root traits (adventitious root number at internodes 7 and
8, and root angle at internode 7) mapped in the SC343B-C403 interval on bin 5.05.
Epistasis was suggested by Guingo et al. (1998) as a possible factor responsible for
the small number of QTLs detected in their study. In fact, the detection of epistatic
interactions requires the evaluation of a much larger set of RILs (Beavis 1994,
1998). A major QTL affecting root traits and root lodging was described by Giuliani
et al. (2005b) and Landi et al. (2005) on bin 2.04. The details for this QTL are
reported in Sect. 8.3.1.
8.3 Production and Characterization of Near Isogenic Lines

for QTLs for Root Traits
For a given genetic background, the accurate characterization of the effects of a

QTL requires the production of near isogenic lines (NILs). Their evaluation will
remove confounding effects on the investigated trait due to unlinked QTLs for
which the parental lines of the RILs may harbour functionally different alleles
(Tuberosa et al. 2002a). A common approach for QTL isogenization relies on the
identification of F4–F5 plants still heterozygous at the target region and their selfing
for a few generations (up to F8–F9) with continued selection for heterozygous plants
before deriving the NIL pairs homozygous and contrasted for the target QTL
interval (Tuinstra et al. 1997). Ideally, the isogenization of a QTL should be carried
out using multiple plants tracing back to different F2 plants. This procedure will
insure a more solid evaluation of the effects of the QTL irrespectively of the
genomic make-up at other QTLs which may influence the target QTL. Alterna-
tively, each parental line of the original mapping population evaluated for discov-
ering the QTL can be used as recurrent parent in a backcross scheme in which a
single plant heterozygous at the QTL in question is utilized as donor of the
alternative QTL regions; in this case, the isogenic lines are identified as back-
crossed derived lines (BDLs; Alonso-Blanco and Koornneef 2000).
Regardless of the method used to obtain NILs, for a cross-pollinated species
like maize that suffers greatly from inbreeding, the evaluation of the effects of a
particular QTL on yield or other highly heterotic traits should preferably be carried
out in a highly heterozygous background. This is usually achieved by crossing the
pairs of NILs with suitable testers. Alternatively, the availability of BDLs allows for
the production of near isogenic hybrids (NIHs) which, depending on the BDLs used
as parents, are either homozygous or heterozygous at the target QTL region, while
being heterozygous for most of the remaining portion of the genome (Giuliani et al.
2005b). Therefore, the evaluation of NIHs as compared to testcrosses allows one to
accurately estimate for both additive and dominance effects of the target QTL.
Major drawbacks to a more widespread utilization of NILs are (1) the specificity
of their effect to a particular genetic background and (2) the long time required for
their production. Commonly, several years elapse from the identification of a major
QTL to its isogenization. This hurdle can be partially overcome with the production
of introgression lines (ILs) involving parental lines preferably contrasted for the
target trait. An IL library is a collection of backcrossed NILs that differ for a small
portion (usually ca. 15–30 cM) of the donor genome. In maize, an adequate
coverage of the entire genome requires ca. 80–100 lines. Once the ILs are made
available, the fine mapping of any major QTL segregating in the original cross can
be readily undertaken (Salvi and Tuberosa 2005). Additionally, the availability of a
collection of ILs allows for testing the presence of epistatic interactions between
specific QTLs. In maize, ILs have been produced in recent years (Szalma et al.
2007; Hao et al. 2009). At DiSTA, we have developed a library of ILs derived from
B73 (recurrent parent) Gaspé Flint (donor parent) to identify major QTLs
influencing, among other traits, root growth and architecture (Ricciolini et al.
2008). Gaspé Flint is an extremely early accession which has been used for the
identification and cloning of early flowering QTL alleles (Vladutu et al. 1999; Salvi
et al. 2002, 2007). A preliminary evaluation of root features in the ILs has allowed
Ricciolini et al. (2008) to identify four bins harbouring QTLs with major effects
on root architecture. The fine mapping of one of these QTLs is underway as a
prerequisite to its positional cloning.
The positional cloning of a major QTL (Salvi and Tuberosa 2005) requires the
availability of (1) a large mapping population (>2,000 plants) derived from the
cross of two NILs for the target QTL, (2) the genomic sequence for the physical
interval spanning the QTL region (obvious starting points are the web-based
genome browser of the target species or at least a contiged genomic BAC library),
and (3) forward- and reverse-genetics approaches for validating the identity and
testing the effects of candidate sequences (coding and non-coding). Only a handful
of the root QTLs reported so far are suitable for a positional cloning approach, the
main obstacle being the vast amount of resources needed to accurately measure
roots in the thousands of plants to be phenotyped in any QTL cloning project.
Additionally, positional cloning in maize is made more complex by its large
genome size and functional redundancy. The availability of the annotated sequence
of the entire maize genome will facilitate the identification of candidate genes and
will streamline the relevant molecular procedures as well as a more effective
comparative analysis with the sequence of other species (e.g., sorghum and rice).
8.3.1 Effects of Root-ABA1 on Root Architecture, ABA

Concentration, Root Lodging, and Grain Yield
In maize, the most extensive evaluation of NILs for root architecture has been
carried out for a major QTL originally mapped for its effects on L-ABA and other
drought-related traits on bin 2.04 in the Os420 IABO78 background (Tuberosa
et al. 1998; Sanguineti et al. 1999). Following the production of NILs (Landi et al.
2005), this QTL was shown to influence root architecture, root lodging, grain yield,
and other important agronomic traits (Giuliani et al. 2005b; Landi et al. 2007). In
this case, backcrossing was used to obtain pairs of BDLs contrasted for the parental
chromosome segments at the target QTL, herein identified as (+/+) and (/) for
their effects on L-ABA (Landi et al. 2005). When the BDLs were tested under both
water-stressed (WS) and well-watered (WW) conditions, the effect of the QTL on
L-ABA was fully confirmed. Subsequently, NIHs for the QTL were developed and
field tested for 2 years under WW and WS conditions. Relative differences among
NIHs for L-ABA and other morpho-physiological traits were not influenced by the
level of water supplied through irrigation (Giuliani et al. 2005b). Interestingly, the
QTL allele for high L-ABA markedly reduced root lodging. To further characterize
the effects of the QTL on root features and L-ABA, plants of two pairs of BDLs
were measured in soil columns at three water regimes. The results confirmed the
effects of the QTL on L-ABA and highlighted a significant effect on several root
architectural features, such as root angle, branching, number, diameter, and dry
weight. Based on these and previously published results, Giuliani et al. (2005b)
postulated a primary, constitutive effect of the QTL on root architecture and size
which, in turn, affects root lodging and also L-ABA. Consequently, the QTL
has been identified as root-ABA1. The QTL allele for a larger and more superficial
root mass was associated with a higher concentration of L-ABA, a finding that
Giuliani et al. (2005b) tentatively attributed to the fact that superficial roots are
more likely to accumulate ABA that is subsequently translocated to the leaves via
xylem flow.
Further validation of the effects of root-ABA1 on grain yield was sought in
different genetic backgrounds. For this purpose, the (+/+) and (/) BDLs were
crossed with five and 13 inbred lines of different origin, thus originating two sets of
testcrosses that were tested in replicated field trials carried out in Italy and China,
respectively, under both WW and WS conditions (Landi et al. 2007). In Italy,
testcrosses derived from (+/+) BDLs were confirmed as less susceptible to root
lodging across both water regimes than the TCs derived from (/) BDLs (28 vs.
53%), but were also lower yielding under WS conditions (4.8 vs. 6.3 Mg ha1). The
testcrosses derived from (+/+) BDLs were also less productive in China (6.8 vs.
7.5 Mg ha1; average of WW and WS conditions). In both sites, the lower grain
yield of the testcrosses derived from (+/+) BDLs was prevalently due to a lower
number of both ears/plant and kernels/plant. These results indicate that the (+) root-
ABA1 allele confers a lower susceptibility to root lodging but also a lower grain
yield, especially in absence of root lodging. The yield loss associated with the (+)
root-ABA1 allele has tentatively been ascribed to the negative effect of an excessive
accumulation of ABA on reproductive fertility (Landi et al. 2007). An alternative
explanation might be that root-ABA1 affects biomass production in response to
drought stress. The fine mapping of root-ABA1 is underway as a preliminary step to
its positional cloning. If successful, the positional cloning of root-ABA1 would
allow us to verify whether pleiotropy or linkage is the prevailing cause of the
multiple effects ascertained for root-ABA1. Additionally, the cloning of root-ABA1
would pave the way to an accurate profiling of elite germplasm to survey the
haplotypes present at the relevant sequence.
Microarray analysis of the transcripts of the contrasting BDLs has been used to
investigate the effects of root-ABA1 on the transcriptome and identify functional
markers tightly linked to the QTL (Giuliani et al. 2005a). This study has led to the
identification of a number of genes preferentially expressed in one of the two BDLs;
among these genes, those which map within the supporting interval of root-ABA1
are being considered as potential candidates for the QTL effects.
8.3.2 Identifying Candidate Genes for Root Features
When a plausible cause–effect relationship can be postulated between a candi-

date gene and an overlapping QTL peak, then validation of the former could be
attempted through genetic engineering and/or the screening of knockout mutants
(e.g., knockouts, TILLING), thus avoiding the time-consuming procedures of the
positional cloning approach. Additionally, the option of the candidate gene
approach can be pursued even with no a priori availability of QTL data. In this
case, association mapping through sequencing or EcoTILLING approach carried
out on a suitable and sufficiently large panel of accessions provides clues on the
association between haplotype variation of the candidate sequence and pheno-
typic variation for the targeted trait. In view of its very low linkage disequilib-
rium, maize is particularly suited for an association mapping approach to validate
the role of candidate sequences. A compelling example of the power of this
approach in maize has been provided by Salvi et al. (2007) through the validation
of the role of a 2.3 kb non-coding sequence that positional cloning in a biparental
background had highlighted as the causative agent of Vgt1, a major QTL for
flowering time.
One merit of the candidate gene approach is that candidates can be identified on
species other than the one being targeted. A clear example in this direction is
offered by several studies conducted in the model species Arabidopsis (Scheres
and Wolkenfelt 1997; Maggio et al. 2001; Flavell 2005; Malamy 2005; Reymond
et al. 2006; Ortega-Martinez et al. 2007; Dello Ioio et al. 2007, 2008; Gonzalez
et al. 2009; Iyer-Pascuzzi et al. 2009) and rice (Ismail et al. 2007; Negrao et al.
2008). In these cases, due appreciation should be given to the fact that the morphol-
ogy and functions of the roots of these species, particularly Arabidopsis, are
considerably different from those of the maize root. Nonetheless, it is possible
that certain core functional/morphological features of root development (e.g.,
signaling cascades, cell elongation, growth and density of root hairs) may have to
a large extent been conserved across species.
The value of using Arabidopsis to elucidate the genetic and functional basis of
root growth has been shown by testing the possible role in root elongation of the
sucrose-splitting enzymes, sucrose synthase and invertase (Sergeeva et al. 2006).
Several QTLs affected both invertase activity and root length. The fine mapping of
a major QTL for root length revealed consistent co-location with the locus for
invertase activity containing a gene coding for a vacuolar invertase. The role of this
invertase gene in root elongation was confirmed by the analysis of a functional

knockout line. Another area worthy of exploration relates to the mechanisms
regulating the level of gene expression in the root. Also in this case, the model
species Arabidopsis has provided useful insights. Although several plant micro-
RNAs (miRNAs) have been shown to play a role in plant development, a study in
Arabidopsis has shown the effect on the root phenotype due to a reduced expression
of a miRNA (Guo et al. 2005a). Arabidopsis thaliana miR164 was predicted to
target five NAC domain-encoding mRNAs, including NAC1, which transduces
auxin signals for lateral root emergence. The results of this landmark study indicate
that auxin induction of miR164 provides a homeostatic mechanism to clear NAC1
mRNA to down-regulate auxin signals; they also show the value of using Arabi-
dopsis as a model for elucidating the complex molecular mechanisms regulating an
important feature of root growth. Genome-wide bioinformatic analysis of full-
length cDNA databases in Arabidopsis has allowed Ben Amor et al. (2009) to
show that the adaptive response of root growth to abiotic stress was controlled by a
long non-protein coding RNA (npcRNA), an emerging class of riboregulators
which either act directly in this long form or are processed to shorter miRNA and
siRNA (short interfering RNA). A number of npcRNAs were antisense to protein-
coding mRNAs, suggesting their cis-regulatory roles. Ben Amor et al. (2009)
proposed npcRNAs as candidate regulators to adapt root growth and development
to soil biotic and abiotic interactions. Nonetheless, the candidate gene approach
suffers from several notable shortcomings which might make its application risky,
particularly with inherently complex traits which are likely to be more “buffered”
from a functional standpoint and, as such, less likely to unequivocally show the
effects of allelic variation at the candidate locus.
8.4 “Omics” of Maize Root Development and Functions
The identification of suitable candidate genes can be facilitated by exploit-

ing platforms that allow us to profile in a high-throughput fashion the trans-
criptome (Schnable et al. 2004; Giuliani et al. 2005a; Guo et al. 2006), proteome
(Hochholdinger et al. 2004a, 2005; Wen et al. 2005; Sauer et al. 2006), and meta-
bolome (Steuer et al. 2003). It should be noted that while microarray platforms
allow for the simultaneous analysis of tens of thousands of transcripts in a single
experiment, or even the entire genome when the relevant sequences are available,
proteomics (Liu et al. 2006) and metabolomics (Fernie and Schauer 2009) can
indirectly report changes occurring in only a tiny portion of the genome. Moreover,
proteomics is often unable to detect the changes in gene products (e.g., transcription
factors) that, despite their low level, can play an important role in root growth and
its response to environmental constraints.
Bruce et al. (2001) were first to deploy a high-throughput approach to investigate
the root transcriptome in two maize lines characterized by contrasting root features.
Among the 13,500 cDNA fragments that were analyzed at two growth stages, 69
showed a twofold or greater difference between the lines at both samplings,

suggesting a relationship between these genes and root anchorage traits.
Because maize roots are composed of different tissues and cell types, each with
its own peculiar signature at the transcript, protein and metabolic level, physical
separation of such cell types can greatly increase our capacity to identify the
specific functions of genes whose activity determines the specificity of root archi-
tectural features. An important breakthrough in this direction has been made
possible through the introduction of laser-capture microdissection (LCM; Schnable
et al. 2004; Balestrini and Bonfante 2008; Nelson et al. 2008), which allows for the
accurate isolation of a wide variety of cell types from complex organs comprising
different cellular types such as the root tip. Transcript profiling of LCM-derived
samples of pericycle and root cap cells in the differentiation zone of primary
roots has unveiled an unsuspected level of functional complexity that would other-
wise have gone undetected (Woll et al. 2005; Jiang et al. 2006; Hochholdinger
et al. 2008).
Transcriptome studies are particularly suited to investigate the adaptive response
of maize roots to environmental cues as evidenced by Liu et al. (2008b) in their
study to investigate the effects on gene expression of local nitrate-induced lateral
root formation in maize. These results showed that local nitrate application induced
the expression of genes related to nitrate uptake and assimilation, sugar transport
and utilization, and cell division and expansion. A similar approach was used by
Spollen et al. (2008) to elucidate the mechanisms underlying the adaptation of
maize roots to low water potential in the elongation zone of maize primary roots
grown under well-watered and water-deficit conditions. This study revealed that the
response to water stress in different regions of the maize primary root involves
different signaling and metabolic response mechanisms. It is worth noting that the
largest functional categories of differentially expressed transcripts were those
related to reactive oxygen species (ROS) and carbon metabolism in root tips and
membrane transport in the elongation zone (Spollen et al. 2008). Microarray
profiling of roots under low-oxygen conditions typically encountered under flood-
ing conditions has shown significant alterations in the expression of 39 miRNAs
(Zhang et al. 2008), several of which targeted transcription factors that were also
induced upon submergence of the maize roots. Other target genes were related
to carbohydrate and energy metabolism, and ROS removal, suggesting that sub-
mergence-responsive miRNAs regulate the adaptive response of maize roots post-
transcriptionally.
New insights into the regulation of maize root development have also
been contributed by proteome profiling studies conducted with complete roots
(Hochholdinger et al. 2004c, 2005; Liu et al. 2006; Sauer et al. 2006; Hoecker
et al. 2008) or targeting more defined sub-cellular portions (Hachez et al. 2006; Zhu
et al. 2006, 2007) of maize roots. Proteome profiling in the elongation zone of the
primary root identified a number of cell wall proteins (CWPs: e.g., endo-1,3;1,4-
b-D-glucanase and a-L-arabinofuranosidase) involved in cell wall metabolism and
cell elongation that had not been previously described in maize (Poroyko et al.
2007; Zhu et al. 2007). Targeting specific cell types via LCM in the primary root of
the mutant lrt1 which is suppressed in lateral roots initiation, Hochholdinger et al.
(2004c) demonstrated the influence of lateral roots on the proteome composition of
the maize primary root. Additional comparative work of the proteome profiles of
primary roots from the wild-type and the rum1 mutant (also suppressed in lateral
root formation) suggested the involvement of post-transcriptional mechanisms in
regulating the mutant phenotype (Liu et al. 2006). Using LCM and combining
microarray profiling with suppression subtractive hybridization, EST sequencing,
and proteomics, Dembinsky et al. (2007) have identified pericycle-specific genes
that appear to be related to the specification of this root cell-type and in lateral root
initiation.
8.5 Conclusions and Challenges Ahead
As shown by this review, genomics allows us to partially dissect the genetic and
functional complexity governing root architecture in maize and its plastic response
to environmental cues. On an adaptive basis, the comparison of transcriptome and
proteome profiles of roots exposed to water deficit (Zhu et al. 2007), water logging
(Zhang et al. 2008), low phosphorous (Li et al. 2007), and low nitrogen (Liu et al.
2008b) has highlighted genes and proteins that might have an adaptive value under
such adverse conditions (Bramley et al. 2007), offering new avenues for more
targeted breeding activities aimed at mitigating the negative effects of environmen-
tal constraints. It is becoming increasingly clear that the response of plant genomes
to environmental stress generates both novel genetic and epigenetic (e.g., methyla-
tion) polymorphisms that may increase phenotypic diversity and plasticity to
abiotic stress (Johannes et al. 2008; Zhang 2008; Chinnusamy and Zhu 2009).
Deep sequencing of cDNA libraries of root cell types will produce extensive EST
databases and unigene sets to identify candidate genes while providing valuable
markers for functional maps (Lister et al. 2009). High-throughput genomic profiling
based on the detection of single nucleotide polymorphisms (SNPs) has vastly
improved our capacity for allele mining (Ganal et al. 2009; Waugh et al. 2009), a
key feature for optimizing the survey of natural variation and the application of
association mapping for complex traits (Weber et al. 2008).
From an architectural standpoint, the cloning of major QTLs will eventually
shed light on the genetic mechanisms governing the quantitative variability of root
structure and its influence on major functions. In this respect, new insights will
derive from a better understanding of the role of miRNAs on the modulation of gene
expression (Sunkar et al. 2007; Ding et al. 2009). Recent experiments have high-
lighted the importance of RNA interference for the regulation of the expression of
genes and QTLs (Guo et al. 2005a; Lukens and Zhan 2007). From an applicative
standpoint, the main challenge remains how to tangibly integrate into extant
breeding programs the deluge of molecular information generated through genomics
and the “omics” platforms. An equally challenging and limiting factor is our
capacity to accurately phenotype roots on the massive scale that genomics studies
usually require (Armengaud et al. 2009). High-throughput phenotyping platforms

(Granier et al. 2006; Rajendran et al. 2009; see also the “Plant Accelerator” at http://
www.plantphenomics.org/TPA) coupled with non-destructive, advanced technolo-
gies promise to alleviate the tedious work of measuring roots, thus opening up new
opportunities to deploy more powerful mapping approaches such as nested-associated
mapping (NAM; Yu et al. 2008).
The need and urgency to fill the genotype-to-phenotype gap (Yano and Tuberosa
2009) has never been more evident than with the study of root architecture, particu-
larly under drought conditions (Tuberosa and Salvi 2006). The limitations inherent to
quantitative trait dissection suggest that only a fraction of the available genotypic
variability will be accessible and amenable to a more direct manipulation via marker-
assisted selection. Even though positional cloning may become a reality for a handful
of major QTLs governing root architecture, the multitude of minor QTLs that control
variability in root features will remain undetected even with the most accurate
phenotyping platforms and sophisticated statistical approaches. Genome-wide selec-
tion bypasses QTL identification (Bernardo and Yu 2007; Bernardo 2008, 2009;
Heffner et al. 2009). Nonetheless, also genome-wide selection relies on accurate
phenotyping which is often considered the main limiting factor for the dissection of
quantitative traits.
Growing attention is being devoted to the opportunities offered by modeling in
order to expand our capacity to predict the effects that specific environmental (e.g.,
water and nutrient availability) and genetic (e.g., QTL effects; Tardieu 2003;
Welcker et al. 2007; Collins et al. 2008; Hammer et al. 2009) variables might
have on plant growth and final yield. Crop modeling has also the potential to help
resolving genotype environment interactions as well as the genetic basis of traits’
plasticity (Chapman et al. 2003; Reymond et al. 2004; Cooper et al. 2009). For
this approach to be effective, crop models that are capable of predicting yield
differences among genotypes in a population under various environmental condi-
tions are needed (Tardieu 2003; Hammer et al. 2005, 2006; van Eeuwijk et al. 2005;
Cooper et al. 2007). The ultimate goal of the modeling approach is to empower
an in silico selection able to pinpoint the combinations of the desirable alleles
at the target loci, including those that dictate root growth and its morphology,
thus providing clues on the desired root phenotype. Clearly, integrative and
interdisciplinary approaches will be instrumental to advance our understanding of
root growth and, eventually, effectively exploit marker-assisted selection and
genetic engineering to tailor root architecture in maize for improving yield and its
sustainability.
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Chapter 9
Phenotyping for Root Traits and Their
Improvement Through Biotechnological
Approaches for Sustaining Crop Productivity
M.S. Sheshshayee, Ehab Abou-Kheir, Sreevathsa Rohini, Namita Srivastava,

B. Mohanraju, Karaba N. Nataraja, T.G. Prasad, and M. Udayakumar
Contents
9.1 How Did the Roots Evolve? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
9.2 Why Are Roots Important for Crop Productivity? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
9.3 Molecular and Hormonal Regulation of Root Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
9.4 Functions of Root in Uptake of Water and Nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
9.5 Nutrient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
9.6 Relevance of Root Traits in Drought Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
9.7 Improving Drought Tolerance Through Exploitation of Root Traits . . . . . . . . . . . . . . . . . . . 214
9.8 Measurement of Root Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
9.9 Oxygen Isotope Ratio as a Surrogate for Root Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
9.10 Genetic Variability in Root Traits and Their Relevance in Improving Crop Growth . . 219
9.11 Breeding for Drought Tolerance Through Root Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
9.12 Transgenic Approach for Root Trait Improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
9.13 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
9.1 How Did the Roots Evolve?
The general structure and function of roots and shoots are so different that the two
organs are often conveniently separated for the purposes of research. Functionally,
roots absorb water and nutrients, and anchor the plant, while shoots photosynthesize
and transpire and are the site of sexual reproduction (Groff and Kaplan 1988). The
exact time when root started appearing has been difficult to ascertain, and the fossil
records are also less helpful for roots unlike shoots. It is possible that delicate
structures such as root caps, root branches, etc. were not properly preserved in fossil
remains (Gensel et al. 2001). Evidences suggested that root-like structures appeared
sometime during the early Devonian period (Elick et al. 1998). Although the early
fossils did indicate the possibility of a root structure positioned to anchor the shoot
M.S. Sheshshayee (*), E. Abou-Kheir, S. Rohini, N. Srivastava, B. Mohanraju, K.N. Nataraja,

T.G. Prasad, and M. Udayakumar
Department of Crop Physiology, University of Agricultural Sciences, GKVK Campus, Bangalore
560065, India
e-mail: msheshshayee@hotmail.com

206 M.S. Sheshshayee et al.
firmly, their role in water- and nutrient-absorption was not clear (Raven and
Edwards 2001). Plants colonizing land must have faced powerful evolutionary
pressures, which must have forced the roots to increase the absorptive surface to
match the development of photosynthetic organs (Brundrett 2002).
In the present scenario, with the continued emission of green house gases, a
definite change in the weather, both locally and globally, is expected. Most predic-
tions suggest that this climate change is inevitable and would lead to a significant
alteration in the pattern and distribution of rainfall in the warmer world (IPCC
report 2007). Hence, water shortage would be the most predominant constraint for
achieving potential productivity of crop plants, especially in tropical regions.
9.2 Why Are Roots Important for Crop Productivity?
Over two-thirds of the world’s human population consumes rice and wheat as staple
cereals, which are predominantly grown under irrigated conditions. With the
changing scenario, it would be difficult to produce the required cereals through
irrigated ecosystems. Furthermore, almost all the pulses, oilseeds, and other crops
are cultivated in dry land conditions, where water is the major constraint. Depen-
dence on dry land agriculture is inevitable in arid and semi-arid tropical parts of the
world. Ironically, these areas are the most populated locations in the world!
Because of the demand from the domestic and industrial sectors, neither expanding
area for agriculture nor finding more water for irrigation would be possible.
Therefore, increasing the productivity per unit of available water appears to be
the only plausible strategy for achieving food security.
Enhancing productivity in the resource-poor dry land conditions is a formidable
challenge. Conserving resources through management practices and engineering
plants for superior extraction of these resources coupled with an increased effi-
ciency of resource utilization deserve emphasis. Though resource conservation
through management practices are equally important, development of superior
resource use efficiency as a seed-based technology always has greater acceptance
and adaptability.
Roots are essential for higher plants for several important reasons. The firm
anchorage of the plant in their soil substratum and the absorption and effective
supply of water and nutrients to the shoot are the most important roles of the root
system. Furthermore, a number of plant growth hormones, especially cytokinins
and ABA, originate in roots, thus having significant influence on growth and
development of plants. Ecologically, roots play a pivotal role in weathering of
rocks, leading to the formation of soil. A mat-like network of root system prevents
soil erosion as well. The evolution of the symbiotic association between roots and
microbes such as Rhizobia, Micorhiza, etc. represents yet another spectacular
feature of plant root system. From the survival and crop productivity point of
view also, roots have a greater role to play. Water mining from deeper soil profiles
is considered as one of the important adaptive strategies evolved by plants to
9 Phenotyping for Root Traits and Their Improvement 207
survive water-scarce conditions. Having realized the importance of root traits in

crop growth and productivity, improving root traits is worth an effort.
In this chapter, we make an attempt to identify a few root-related traits and
review the information describing the relevance of root traits in determining crop
growth and productivity, especially under water-limited conditions. Since the major
emphasis is on breeding for root traits, we also review the genetic variability in root
traits and describe suitable methodology for the assessment of variations in root
traits. In the present scenario, greater success in crop improvement can be achieved
only through a trait-based approach. Introgression of complex traits is best achieved
either by a well-focused molecular breeding strategy or through transgenic technol-
ogy. A better understanding of the basic mechanisms of root growth and develop-
ment is necessary for these modern biotechnological approaches to become
successful.
9.3 Molecular and Hormonal Regulation of Root Growth
In both the dicot and monocot plant species, the short-lived delicate roots perform
the function of absorption, while the more long-lived roots help in anchoring the
plants. The basic features of root development has been analyzed by dividing the
root tip into different parts such as root cap, the meristematic zone, elongation zone,
and maturation zone. Root growth occurs due to the division, elongation, and
differentiation of the root apical meristematic cells present in the root tip. The
lateral growth of roots occurs only after the complete elongation of apical meristem
and at a distance away from the root tip (Malamy and Benfey 1997). The pattern of
the root growth is strongly controlled by both external and internal factors. While
external factors such as soil structure, availability of water and nutrient, etc.
determine root growth and patterning, the internal factors are predominantly
under hormonal control, which determine the plant’s ability to respond to the
external stimuli. The internal control of root growth by genes and their regulatory
network in root development is partly examined in Arabidopsis through global gene
expression studies. Many mutants that affect root development have also been
identified and characterized, which has led to a clear understanding of the genetic
mechanisms of root development (Schiefelbein 2003; Casimiro et al. 2003; Casson
and Lindsey 2003; Inukai et al. 2005). These efforts resulted in the discovery of a
large number of structural and regulatory genes. Several of the regulatory genes,
also referred to as Transcription factors (TFs), have been cloned and characterized
and their functional relevance clearly demonstrated. A few of the important genes
and their regulatory functions are summarized in Table 9.1.
Further, plant roots show an impressive degree of plasticity in adapting their
branching patterns to the ever-changing growth conditions. The adaptation ability
depends upon the interaction between hormonal, developmental, and environmental
signals. Root growth and development is also influenced by hormones. Research
reports accruing in the recent years point towards auxin as one of the prominent
Table 9.1 Some important transcription factors (TFs) and their role in root growth in plants
Transcription factor Phenotypes Reference
SLR/IAA14 Blocks lateral root formations in Fukaki et al. (2002)
Arabidopsis
CRL1 Encodes for protein family that govern Inukai et al. (2005)
asymmetric leaves/lateral organ
boundaries. Positive regulator for
crown and lateral root formation
ARL1 Encodes lateral organ boundaries Liu et al. (2005)
(LOB); adventitious root formation
NAC1 More lateral roots Xie et al. (2000, 2002)
Class III HD-Zip Promote the meristematic activity, Hawker and Bowman (2004)
positive regulators of lateral root
formation
SCR-SCARECROW Auxin responsive: organization and Wysocka-Diller et al. (2000),
SHR-SHORT-ROOT quiescent center cells and root cap Gao et al. (2004),
Helariutta et al. (2000)
Alfin1 Over expression enhances root growth Winicov (1993, 2000),
under normal and saline conditions Bastola et al. (1998),
in Alfalfa Winicov and Bastola
(1999)
OsRAA1 Root development in rice initiation and Ge et al. (2004)
growth of adventitious roots
Ca2+-dependent protein Regulates diverse processes including Ivashuta et al. (2005)
kinase1 (CDPK1) root growth
CAP2 encoding Over expression of chickpea CAP2 Shukla et al. (2006)
APETALA2 (AP2) caused drastic increase in the
number of lateral roots
HARDY (AP2-family) Better root growth and drought Karaba et al. (2007)
tolerance
ARABIDILLO 1 and 2 Armadillo-related b-catenin-like Coates et al. (2006)
proteins Over expression increased
lateral root formation
QHB (QC SPECIFIC Maintenance of root meristem by Kamiya et al. (2003)
Homeodomain) inhibiting the differentiation of the
adjacent initial cells
MYB77 Controls lateral root growth through Shin et al. (2007)
interaction with Auxin response
factor (ARF)
KNAT6 Member of the knotted-like (KNOX) Dean et al. (2004)
gene family. Prevent production of
supernumerary lateral roots
internal controlling factors of root growth (Xu et al. 2005; Lucas et al. 2008). The
process of root development can be divided into two successive stages: lateral root
initiation and lateral root development/emergence. Auxin controls the emergence of
lateral root primordia and also helps in the growth and development of lateral roots
(Bhalerao et al. 2002; Casimiro et al. 2001; De Smet et al. 2007; Fukaki et al. 2007).
There are experimental evidences to show that the number of lateral roots can be
altered either by application of auxin or perturbation of internal auxin levels (Blakely
and Evans 1979; Blakely et al. 1988). The hormone is synthesized mainly in the young
apical tissues and then transported downwards to different parts, including roots, by a
polar transport system (Muday and DeLong 2001). A number of auxin influx carriers
(e.g., AUX1 and LAX gene family) and efflux carriers (e.g., PIN gene family) have
been characterized (Bennett et al. 1996; Friml et al. 2002; Reinhardt et al. 2003) and
relevance of such auxin-related genes in root development has been demonstrated. For
example, Arabidopsis mutant called pin-formed (pin1) fail to establish endogenous
auxin gradient and show development disorders in root (Okada et al. 1991; Benková
et al. 2003). A gene similar to Arabidopsis PIN1 has been identified in rice (OsPIN1),
which, through transgenic approach, has been implicated for altering tiller number
and adventitious root development in rice (Xu et al. 2005).
Though a clear implication of auxin is seen in the root growth and development,
understanding of the molecular basis of this regulation is not complete (Weijers and
Jurgens 2005). However, investigative evidences accruing in the literature have
provided significant lead towards understanding the response of root growth to
auxin in plants. Transcription factors (TF) called auxin-response factors (ARFs)
bind to auxin response elements (AuxREs) in the promoters of auxin-response
genes to mediate auxin-induced responses (Ulmasov et al. 1997). The auxin recep-
tor TIR1 (F-box protein), which acts by mediating the degradation of AUX/IAA
(AUXIN-RESPONSIVE PROTEIN/INDOLE ACETIC ACID INDUCED PRO-
TEIN) repressor is the most important member involved in the auxin response
process promoting the lateral root initiation (Gray et al. 1999, 2001). The F-box
gene called CEGENDUO (CEG) negatively regulates auxin-mediated lateral root
formation, which is expressed abundantly in vascular tissues of the primary root and
is induced by auxin (Dong et al. 2006).
Interaction of growth regulator jasmonate with auxin to regulate lateral root
formation has been recently reported (Sun et al. 2009) by characterizing an Arabi-
dopsis mutant called jasmonate-induced defective lateral root1 (jdl1/asa1-1). The
JDL1 encodes the auxin biosynthetic gene ANTHRANILATE SYNTHASE alpha1
(ASA1), which is required for jasmonate-induced auxin biosynthesis and affects
auxin transport (Sun et al. 2009). Jasmonate also has a role in the attenuation of
auxin transport in the root and the fine-tuning of local auxin distribution in the root
basal meristem.
Cytokinin suppresses the growth of roots as reported in Arabidopsis (Werner
et al. 2001) by reducing the size and cell division of roots. The roots of cytokinin-
deficient (AtCKK1) plants were larger than those of wild-type, suggesting that the
hormone inhibits root growth. Although most studies have reported genes that are
directly associated with auxin in root development, a few indicate auxin-indepen-
dent mechanisms. For instance, a novel gene called ALF4, which appears to be
localized in the nucleus, was demonstrated to be required for lateral root formation
(DiDonato et al. 2004). The ALF4 functions independent of auxin signaling and has
a role in maintaining the pericycle in the mitotically competent state required for
lateral root formation.
Most other plant hormones seem to have an indirect effect on root growth
through their independent effects on auxin synthesis, transport, and distribution.
For instance, ethylene regulates growth through effects on auxin biosynthesis and
auxin distribution through altered transport. ABA, an otherwise growth-retarding
hormone, promotes root growth possibly by inhibiting ethylene production (Saab
et al. 1990; Sharp 2002).
Genomic approaches have therefore provided immense information about the
array of structural and functional genes involved in various aspects of root growth,
development, and water and nutrient uptake. Use of these genes in overexpression
studies would help validate the utility of these in root trait improvement.
9.4 Functions of Root in Uptake of Water and Nutrients
Other than anchorage, the next important function of roots is to take up water and
nutrient. This trait of roots enables the plants to tide through different environmen-
tal conditions. Terrestrial plants are constantly exposed to an impinging heat load
because of the incident solar radiation. To cope with this heat load and to maintain
the canopy cool, plants transpire enormous amount of water. Plants recycle over
half the amount of global precipitation per annum (Chahine et al. 1992). Hence, the
roots must be able to extract water from the soil and supply it to the plant to match
the evaporative demand of the canopy.
Vascular tissues and guard cells are mainly involved in conducting water and
controlling the transpiration stream. During this, water has to flow in and out of the
cells. This flow of water can be across cell walls (apoplastic path), between cells
across plasmodesmata (symplastic path), or traversing cell membranes (transcellu-
lar path). A better understanding of the conductance of living cells has come from
the discovery of a class of water channel proteins called “Aquaporins” (Agre et al.
1998). These are proteins embedded in the cell membrane and regulate the flow of
water. These aquaporins are integral membrane proteins belonging to a family of
major intrinsic proteins (MIP) that form channels in the membrane for water
movement. More than 50% of the water moving across plant cells would traverse
aquaporins.
In plants, aquaporins are divided into four subfamilies:
1. Plasma membrane intrinsic protein (MIP)
2. Tonoplast intrinsic protein (TIP)
3. Nodulin-26 like intrinsic protein (NIP)
4. Small basic intrinsic protein (SIP)
However, all the aquaporins have six membrane spanning domains with highly
conserved Asn-Pro-Ala motif.
Aquaporins may be involved in a large number of physiological functions in
plants such as response to drought or salinity, mineral nutrition, transpiration, cell
elongation, etc. (Maurel and Chrispeels 2001). The discovery of aquaporins has
showed the importance of membranes in plant–water relations. Further, aquaporins
serve as spatial markers to explore the flow of water and solutes that play a
phenomenally important role throughout plant development.
Besides enhancing water uptake, aquaporins also contribute significantly to the
total hydraulic conductivity of the roots. A significant reduction in water flux
through membranes in the presence of HgCl2 and its reversal with the removal of
mercury by an excess of mercaptaethanol provided the initial proof to the involve-
ment of aquaporins to the hydraulic conductivity of roots in tomato (Maggio and
Joly 1995). Although transpiration pull sufficiently explains water uptake and
distribution in plants, the hydraulic conductivity, the ease with which water
moves through the roots, is an equally important factor. The fact that hydraulic
properties of roots vary with plant species and environmental conditions has been
well-known from a very long time (Brewig 1937; Brouwer 1954). Several factors
influence the hydraulic conductivity of plant, viz. number of roots that are absorb-
ing water (Vandeleur et al. 2005), nitrate nutrition (Radin and Boyer 1982), and
ABA (Hase et al. 2000).
During evolution, plants have also optimized hydraulic conductivity to enhance
their chances of survival under dry and harsh conditions. The evidences to this view
were provided recently by Zhao et al. (2005) using wheat lines with different
ploidy. They clearly demonstrated that root hydraulic conductivity significantly
increased as ploidy level increased during wheat evolution. Since hydraulic con-
ductivity was positively related to plant biomass, the authors opined that increasing
water flux into the shoot would enhance photosynthetic efficiency leading to an
increase in water use efficiency.
9.5 Nutrient
The proper development of roots at all stages will have profound effects on root
system architecture as well as nutrient acquisition. The development of roots is
particularly sensitive to the changes in the internal and external concentrations of
nutrients. Recent information points to the existence of nutrient-specific signal
transduction pathways that interpret the external and internal concentrations of
nutrients to modify root development. Progress in this field has led to the identifi-
cation of regulatory genes that play pivotal roles in nutrient-induced changes in root
development (Lopz-Bucio et al. 2003).
Nitrogen is an important and critical nutrient that determines crop growth and
productivity. For plants, nitrate is the most preferred form of nitrogen and is taken up
by active transport through the roots. Changes in nitrate availability has been found
to have contrasting effects on lateral root formation and elongation (Zhang and
Forde 1998), which is suppressed by both high nitrate and high phosphate availabil-
ity. Some of the components of the signaling pathways that regulate root-system
architecture in response to nutrient availability have been identified. In Arabidopsis,
the NITRATE-REGULATED1 (ANR1) gene encodes a nitrate-inducible MADS-box
transcription factor whose role is speculated in root plasticity in response to nitrate.
In another scenario, crosstalk was found to exist between nodulation and lateral
root development in Lotus japonicus. It was found that HAR1, which encodes a
putative serine/threonine receptor kinase that had homology with CLAVATA1,
was involved. HAR1 is required for shoot-controlled regulation of root growth,
nodule formation, and nitrate sensitivity of symbiotic development (Nashimura
et al. 2002).
Phosphate is one among the least-available macronutrients required by plants
and is a constituent of key molecules such as ATP, nucleic acids, and phospholi-
pids. Phosphate deficiency limits plant growth and development, resulting in
adaptive stress responses. Over the past decade, many genes including phosphate
transporters, phosphatases, RNases, and others of unknown function that help
plants adapt to Pi stress have been characterized. SIZ1, a SUMO E3 ligase, was
identified to control Pi homeostasis at the posttranslational level through sumoyla-
tion (Miura et al. 2005). Earlier, Phi-2, coding for a bZIP transcription factor in
tobacco was reported to be induced during Pi starvation (Sano and Nagata 2002).
Another transcription factor, PHR1, was first reported to play a regulatory role in Pi
starvation responses in Arabidopsis (Rubio et al. 2001). Similarly, tolerance to
phosphate starvation in rice was brought about by OsPTF1, a bHLH transcription
factor (Yi et al. 2005). Very recently, the role of WRKY75 in regulation of Pi
starvation responses in Arabidopsis was evaluated (Devaiah et al. 2007a). To
continue the growing evidence that transcription factors are key components of
nutrient regulation, ZAT6 (zinc finger of Arabidopsis 6), a cysteine-2/histidine-
2 zinc finger transcription factor, is induced during Pi starvation (Devaiah et al.
2007b).
Sulfur is another nutrient important for plant growth. Under deprived sulfur
conditions, plants develop a branched root system. This has been related to the
transcriptional activation of the NITRILASE3 (NIT3) gene, a member of nitrilase
gene family. It is suggested that NIT3 plays direct role in auxin synthesis and root
branching.
Optimum uptake of nutrients from the soil is a very important aspect of nutrient
use efficiency. For this, plants require specialized transporters that are at the root/
rhizosphere interface to take up nutrients. These comprise of high and low affinity
transporters, which allow the plants to transport nutrients from soil to plant.
This need of quenching nutrients make plants to modify their organ development
to enhance their ability to capture water and nutrients. Many species have evolved
mechanisms that allow them to detect nutrient-rich patches in the soil (Zhang
and Forde 1998). In Arabidopsis, nitrate transporter, NRT1.1 has been identified
(Remans et al. 2006). It is seen that NRT1.1 is a key component of the nitrate-
sensing system that enables the plants to detect and exploit nitrate-rich soil patches.
Likewise, transporters have been identified for phosphate as well. Two different
families of transporters have been identified, viz. PHT1 (Liu et al. 2008) and
PHT2 (Versaw and Harrison 2002), which influence the allocation of phosphate
within the plant under phosphate starvation. More recently, another transporter
has been identified recently in Arabidopsis called the PHT64;6, which belongs
to the family of permeases and is found to be a determinant of salt tolerance
(Cubero et al. 2009). Similarly, a high affinity sulfate transporter has been identified
in Arabidopsis thaliana called the HstAt1.
9.6 Relevance of Root Traits in Drought Tolerance
Among a number of stresses that affect crop growth and productivity, drought is
perhaps the most prominent stress. A yield loss ranging between 20 and 60% is
generally noticed in tropical regions. Hence, improving drought tolerance in crop
plants is one of the most essential trusts in global research.
Drought tolerance is the ability of a plant to “avoid” the buildup of stress or to
“tolerate” stress effects at the organism level (Levitt 1972; Blum 2005). However,
improvement in drought tolerance has largely remained academic owing to the
complexity of drought stress and equally complicated crop response to drought.
Past research experience point strongly towards trait-based breeding, notwithstand-
ing the significant progress made by selecting for high yield under water limitation.
Avoidance of stress through conservation strategies such as rapid physiological
development, sensitive stomatal behaviour, heleonastic movements leaves, etc.,
though relevant, are normally counter productive. Ability of the plant to explore
water source and extracting water from deeper profiles of soil thus has great
relevance in maintaining water relation as well as carbon assimilation.
Deep-rooted plants have been shown to be better productive under water-limited
conditions (Li et al. 2005; Reynolds and Tuberosa 2008). Such a trend was recently
noticed also in C4 crop such as finger millet at our centre (Fig. 9.1). Several of the
root-related traits described above have been shown to be related with improved
growth under stress.
Hence, improving these component traits has significance in sustaining produc-
tivity under water-limited conditions. After having achieved considerable under-
standing of root growth and development both at the whole plant level and at the
molecular level, strategic approaches for crop improvement can be formulated.
Trait improvement can be effectively achieved either by introducing validated
genes through transgenic technology or by introgressing desirable alleles through
molecular breeding approaches. The traits relevant for drought tolerance and
productivity are highly species-specific. While the distance from transition zone
to first main lateral root, tap root weight, rapidity of root system development, and
root to shoot ratio are important for cotton’s (Cook 1985; Pace et al. 1999) ability to
penetrate hard pan, root length, basal thick mass, and deep root biomass are
important for rice and wheat.
Though deep-rooted plants produced more grains under low water availability,
these plants had the risk of exhausting soil water early. Hence, Condon et al. (1993,
2004), Richards et al. (2002), and Sheshshayee et al. (2003) have emphasized that
soil factors also need to be considered before attempting to improve root traits.
Despite the realization of the relevance of root traits in imparting drought
tolerance and a good understanding of the molecular mechanisms of root growth,
Fig. 9.1 Differences in total biomass of Finger millet accessions differing in root traits grown
under well watered and water limited conditions. Note: Stress was imposed by gravimetric
approach and maintained for a period of 45 days between 30 and 75 days after sowing.
Source: Shankar (unpublished data)
a thorough exploitation of root traits has not been successfully achieved. Accurate
measurement of root traits is one of the most significant constraints in crop
improvement programs.
9.7 Improving Drought Tolerance Through Exploitation

of Root Traits
O’Toole and De Datta (1986) suggest that drought is a syndrome because of the
uncertainty of its occurrence, duration of its persistence, and the intensity. The soil
characteristics, the crop species, and stages of crop growth all further complicate
the process of understanding drought tolerance. Therefore, addressing drought
tolerance requires a very comprehensive approach.
From the physiologist’s perspective, plant water relations play a very curial role
in determining the level of drought tolerance in plants. Root once again occupies
the pivotal position through its role in extracting water from deeper soil profiles.
Maintenance of tissue water status is linked with (a) better extraction of water
through deep root systems and (b) better water conservation strategies associated
with sensitive stomatal behavior and deposition of waxes on the cuticular surface
(O’Toole and Chang 1979; Ludlow 1993; Ingram et al. 1994). Though the conser-
vation strategies are very useful under water-limited conditions, most of these traits
are counterproductive. Agronomically, any drought tolerance trait would be rele-
vant only when they are also associated with better growth and productivity. Simple
growth models provide the framework for identifying such traits, and one such
model was proposed by Passioura (1986). As per this model, yield of any plant is
a fraction of the amount of water used, the efficiency of water use for biomass
production, and the partitioning of biomass to harvestable parts. Hence, root
traits are strongly associated in enhancing crop productivity under water-limited
conditions.
Significant developments have been achieved in understanding the physiology
of drought resistance and developing physiological screening techniques for
drought resistance, which reduce time in selection programs (Blum 1988; Ludlow
and Muchow 1990). In recent reviews, Sheshshayee et al. (2003) and Reynolds
and Tuberosa (2008) have discussed various traits that deserve exploitation to
achieve drought tolerance coupled with sustained productivity under water-limited
conditions.
A root system that extends the root zone to fully extract available soil water has
the potential to increase yield under drought (Mambani and Lai 1983). Water
uptake and transport by rice roots are most important as they affect yield, especially
under water-limited conditions (Ingram et al. 1994). Individual root characteristics,
such as thickness, depth of rooting, and the ability to penetrate compacted soils,
have been associated with drought avoidance (O’Toole and Chang 1979; Yoshida
and Hasegawa 1982; Ekanayake et al. 1985). Significant genetic variability in some
of these root traits have been demonstrated and implicated for improved drought
tolerance in crop plants (O’Toole and De Datta 1986; Thangaraj et al. 1990; Sharma
et al. 1994; Sinclair and Muchow 2001). Biomass accumulation in plants is always
a function of total water used (Angus and Van Herwerdeen 2001: Passioura 1986).
Plants with deep root system hence have the ability to supply water to support a
higher transpiration demand, thereby enhancing total biomass (Yadav et al. 1997;
Li et al. 2005). In their simulation experiments, Sinclair and Muchow (2001)
demonstrated an increase in biomass and yield when root growth was better.
These studies emphasized the relevance of breeding to improve root traits to
achieve better productivity under water -limited conditions (Reynolds et al. 2007;
Reynolds and Tuberosa 2008).
Besides the inherent genetic variability among most plant species in root traits,
roots are quite dynamic in responding to both biotic and abiotic stresses as well as
soil characteristics. An increase in root length when plants are stressed for water
and for nutrients is well known (Pace et al. 1999). However, when stress levels
become severe, a significant reduction in root growth becomes inevitable (Prior
et al. 1995; Plaut et al. 1996). Variation among genotypes for shifting root distribu-
tion downwards in response to drought has been found in cowpea (Matsui and
Singh 2003), white clover (Annicchiarico and Piano 2004), and chickpea (Yusuf
et al. 2005; Benjamin and Nielsen 2006; Kashiwagi et al. 2006). Absence of
suberized hypodermis would permit rapid desiccation of delicate roots, leading to
an increased root mortality in drying soils (Shone and Flood 1983; Jupp and
Newman 1987; Smucker et al. 1991). A plant’s root growth and extension decrease
as the soil strength increases. Soil strength greater than 0.3–0.5 MPa and soil
bulk density greater than 1.5 g cm3 hamper root growth and penetration below
10–15 cm from the soil surface (Hasegawa et al. 1985; Thangaraj et al. 1990).
Presence of compacted soil layers acts as physical and physiological constraints to
overall plant growth (Tu and Tan 1991) and impede the downward growth and
distribution of the plant root system (Yu et al. 1995). Compacted soil layers reduce
leaf area, dry matter accumulation, root elongation rate, transpiration rate, and crop
yields (Masle and Passioura 1987; Assaeed et al. 1990; Ludlow et al. 1989; Masle
1992). Mechanical disruption of the compacted soil layers has been done to
increase yield in cotton (Camp et al. 1984) and soybean (Khalilian et al. 1991).
But mechanical disruption is expensive, and compacted layer often reform in a few
years (Busscher et al. 1986).
9.8 Measurement of Root Traits
Determination of genetic variability in root traits represents the most difficult

challenge in crop improvement programs. Despite the undeniable importance of
root traits in better water mining, progress in breeding for these traits has been
extremely slow. A few important root-related traits have been enumerated that have
direct relevance for maintaining the balance between water relations and carbon
assimilation of the plant. Several methodologies have also been developed and are
being adopted for studying these root traits.
The simplest and more frequently used method is Hydroponics (Martinez et al.
1998; Tuberosa et al. 2002). This method involves raising of plants in suitable tanks
filled with a nutrient solution. This system provides a very convenient approach for
assessing the variability in root traits. However, lack of proper aeration to the roots is
one of the major disadvantages of this method. Further, large-scale screening of
germplasm and breeding lines would be very tedious. Growing plants in mini-
rhizotrons (Drouet et al. 2005) or mini-lysimeters (Udayakumar et al. 1998) has
also been extensively used for root studies. The rhizotrons can be made of transparent
material that readily allows the direct visual monitoring of root growth and pattern-
ing. Though very effective, extending this method for large-scale screening is quite
difficult. Scientists attempted to raise plants in tubular containers of varying lengths.
These tubes are split in half at the time of harvest and the soil is washed off carefully
to obtain the entire root mass. Normally, one plant is maintained in each container
(Venuprasad et al. 2002; Ayyappa 2004; Giuliani et al. 2005). Plants grown in tubes
can also be used for what is often known as “core-break” technique (Taylor et al.
1991). The soil core is taken completely from the tubes and cut into sizes as desired.
Each of these pieces is then washed and the roots are taken out carefully.
A few destructive sampling techniques are also being adopted for root trait
studies where the root mass is entirely excavated from the soil. Though this method
is quite convenient, it is very difficult to completely excavate the roots, and hence
this method has a significant random error, which would hamper accuracy of
measurements. More sophisticated techniques of determining root growth have
also been developed. A capacitance-based method has been used for monitoring
root growth (Van Beem et al. 1998). This method demands wetting of the soil at
least up to field capacity and hence cannot be effectively used in assessing root
growth in soils with water deficit. X-ray imaging and light transmission imaging are
also being adopted for root trait measurements. A more recent technique of scan-
ning the root system was evolved for cotton, where sampling was done for every
10 cm row for a depth of 0–100 cm of soil using a root sampler. The sampled soil
was washed and scanned using a hand scanner at 200 dpi. The resultant image was
analyzed using a DT-SCAN software (version 1.0; Delta, Inc., UK) to measure root
length, average diameter, and surface area (Bouma et al. 2000; Zhang et al. 2005).
Most of the techniques available for root measurements suffer either due to the
cumbersomeness of the procedure or due to their inability to screen large number of
accessions. Further, root studies using pipes or mini lysimeters do not present the
correct phenotypic expression of the root traits as they do not experience interplant
competitions. Because the space provided in pipes directs more of a longitudinal
growth, lateral root development gets constrained.
Most of these disadvantages can largely be overcome by raising plants in specially
constructed “root structures” (Fig. 9.2). Although various dimensions can be
adopted, the most suitable would be 5-ft tall, 10-ft wide, and 60-ft long structures
built using cement bricks (Fig. 9.2). An additional 5-ft tall wall can be built in the
middle of the structure to make two halves, each 5-ft wide, which provide additional
strength to the structures. Soil is filled in these structures and compacted to mimic the
real field conditions. Crop can be sown or planted in rows, and an exact plant
population can be maintained. This approach provides the near-natural condition
for phenotyping. Since the plant population is maintained as that in the main field, the
plants would experience the interplant completion, which might have an important
effect on the phenotypic expression of root growth. Thus, the measurements of the
root traits from plants grown in such root structures would be very accurate. At the
end of the experiment, the brick walls along the sides can be dismantled with care and
the soil washed away using a strong jet of water. The roots are separated carefully
Fig. 9.2 Specially constructed root structures to assess genetic variability in root traits in large
number of accessions. Note: Each of these structures measure 60-ft long, 5-ft tall and 10-ft wide
and is constructed using cement bricks. A wall is built in the center for dividing the structures into
two halves of 5-ft wide each
from soil particles and then used to record various parameters such as root length,
number of primary and secondary roots, root volume, etc. The roots can then be
separated from the shoots and oven dried to measure root biomass. Except for the fact
that the plants are grown in raised structures, this approach provides an option for
determining genetic variability among large number of accessions in several root
traits in conditions that are almost natural.
9.9 Oxygen Isotope Ratio as a Surrogate for Root Traits
Alteration in the stable isotopic composition of water has been well known to occur
during evaporation. Although the theory explaining the phenomenon of oxygen
isotopic enrichment during evaporation of water from ocean surface has been
known for almost four decades (Craig and Gordon 1965), the application of this
theory to predict differences in transpiration rate has been fairly recent (Flanagan
et al. 1991, 1994; Farquhar and Lloyd 1993; Bindumadhava et al. 1999). However,
discrepancy between the Craig-Gordon prediction and the measured d18O of the
leaf water has been reported (e.g., White et al. 1994; Buhay et al. 1996). Further, the
relationship between stomatal conductance and leaf water 18O enrichment has
remained equivocal (Farquhar et al. 2007), though increased transpiration has
been clearly shown to enrich leaf water 18O (Gonfiantini et al. 1965; DeNiro and
Epstein 1979). We recently provided experimental evidences and demonstrated that
oxygen isotope enrichment is a powerful time-averaged surrogate for transpiration
rate (Fig. 9.3) (Sheshshayee et al. 2005)
A 39
37
35
D 18O1b
33
31
29
27
150 170 190 210 230 250 270 290
MTR
Fig. 9.3 Relationship between oxygen isotope enrichment (D18O) and transpiration rate in rice
genotypes (from Sheshshayee et al. 2005)
Fig. 9.4 Oxygen enrichment 20
accurately reflects root y = 2.475x –79.52

biomass in rice. Note: root 16
R2= 0.828
traits were measured by
Root wt (g l–1)
12
growing contrasting
genotypes in root structure.
8
Leaf samples were taken for
the determination of oxygen
4
isotope ratio using IRMS at
department of Crop 0
Physiology, UAS, Bangalore 31 32 33 34 35 36 37 38 39
(Mohankumar and δ18O(permill)
Sheshshayee – Unpublished
data)
In most plant species, the total biomass accumulated is a function of the total
water used through transpiration. Total transpiration is further a function of the
evaporating surface area of the canopy and the extent of root development to supply
water to match the evaporative demand. Hence, transpiration at a given leaf area
must be related to root biomass and hence a good indicator of root traits. Oxygen
isotope enrichment values at a given leaf area was found to be strongly correlated
with root biomass in both annual and perennial crop species (Fig. 9.4). Being high
throughput and very accurate, stable isotope ratio is a very useful approach for the
determination of root traits in plants.
9.10 Genetic Variability in Root Traits and Their Relevance

in Improving Crop Growth
Success in breeding for any trait entirely depends on the existence of exploitable
genetic variability in that trait with moderate to high heritability. Research on the
genetic variation and heritability of rice root traits was reviewed by O’Toole and
Bland (1987). Geneticists have estimated both broad sense and narrow sense herita-
bility for root traits and have reported additive gene action and polygenic inheritance
of most root traits. Maximum root length, root diameter, root dry weight, root length
density, root number, root–shoot ratio, root dry weight, thick root number below
30 cm, and root mass density at different depths of soil layers are a few such traits.
Significant genetic variability in these traits has also been reported (Chang et al.
1982; Armenta-Soto et al. 1983; Sharma et al. 1994; Ekanayake et al. 1985).
These traits have significant implication to the growth of plants, especially under
water limitation. Root morphology and rooting patterns directly influence the
amount and timing of water supplied to the crop canopy (Champoux et al. 1995).
Root penetration ability associated with a few thick and long root axes helps to
penetrate the compacted soil layer to reach the water source (Yoshida and
Hasegawa1982; Ekanayake et al. 1985; Ingram et al. 1994; Yu et al. 1995;
Zheng-Xiang et al. 1998). These long roots through efficient absorption of water
have a positive influence on biomass (Yadav et al. 1997). Longer and larger roots
have wider xylem diameter, thus contributing to higher hydraulic conductivity and
better water uptake (Passioura 1982; Ludlow and Muchow 1990). Variability in
root traits among a few important crop species was noticed in various experiments
conducted at our center. The mean and range of a few important root traits are
indicated in the Table 9.2.
Similarly, several workers reported exploitable genetic variability in many root-
related traits (Cook 1985; Pace et al. 1999; AbouKheir et al. 2008) and demon-
strated variation in root hair density, hydraulic conductivity, and root length density
in chickpea (Kashiwagi et al. 2005, 2006); analysis of the genetic control of these
traits has revealed a multigenic inheritance with a predominance of additive gene
action. Most of the research on assessing root traits has concentrated on cereals like
rice and wheat. Price et al. (1997), while examining both Indica and Japonica
varieties of rice, showed a significant additive and dominance effects on several
root-related traits.
9.11 Breeding for Drought Tolerance Through Root Traits
Despite the realization of the importance of roots in crop growth and productivity,
especially under water-limited conditions, no serious breeding efforts have been
initiated till date to improve root traits (Blum 2005). Lack of a proper phenotyping
strategy for root traits is perhaps the most important constraint. Though several
techniques have been developed and are being used to assess root traits, screening
large number of accessions is still a major challenge.
Among all the different abiotic stresses, drought is the most complex and devas-
tating on a global scale (Pennisi 2008). Hence improving drought tolerance of crop
plants deserves the greatest emphasis. However, progress towards this endeavour has
been show primarily because of an ambiguous definition to drought and drought
tolerance in the literatures (Tardieu 2003; Blum 2005; Collins et al. 2008). Remark-
able dehydration tolerance has been achieved by adopting genetic engineering
strategies that have targeted improvement in a range of processes including cell
protection mechanisms (Jenks et al. 2007; Nelson et al. 2007), detoxification of
reactive oxygen species that accumulate under stress (Lee et al. 2007; Yang et al.
2007), and hormonal manipulations that regulate adaptive strategies (Rivero et al.
2007). Nevertheless, these approaches have only provided drought tolerance in a
laboratory condition while having little yield advantage under a much milder and
intermittent drought conditions that are normally encountered in commercially
cultivated field conditions (Collins et al. 2008). In contrast, exploration of natural
variation in drought-related traits has resulted in a slow but a definite progress in crop
performance (Rebetzke et al. 2002; Ribaut et al. 2004; Reynolds and Tuberosa 2008).
Table 9.2 Genetic variability in a few root traits in several crop and perennial species
Crop species Trait Minimum Maximum Mean SD Source
Finger millet (n ¼ 270) Root wt 2.12 19.92 9.26 3.41 Rajashekar Reddy and Shankar (personal communication)
TDM 50.25 125.7 47.45 14.12
R/S ratio 0.09 0.66 0.25 0.09
Rice (n ¼ 230) Root wt 1.83 17.6 8.61 3.3 Mohan Kumar and Sheshshayee (unpublished data)
TDM 12.68 84.10 40.68 14.45
R/S ratio 0.1 0.75 0.28 0.12
Sunflower (n ¼ 140) Root wt 5.47 137 39.8 29.5 Vikarm and Mohan Raju (personal communication)
Root length 20.33 81.25 43.78 11.88
Root volume 20 335 112.7 63.9
TDM 85.8 357 184.4 64.3
R/S ratio 0.068 0.67 0.26 0.16
Groundnut (n ¼ 260) Root wt 0.85 3.9 1.9 0.49 Namita and Sheshshayee (unpublished data)
Root length 28.7 76.5 48.1 8.3
TDM 9.4 97.10 37.8 12.3
R/S ratio 23.9 123.0 54.4 14.1
9 Phenotyping for Root Traits and Their Improvement
Mulberry (n ¼ 175) Root wt 6.53 113.9 39.0 19.2 Vinoda and Sheshshayee (unpublished data)
Root length 35 116 71.3 14.9
Root volume –
TDM 39.10 356.20 207 31.0
R/S ratio 0.25 0.42 0.38 0.43
Cotton (n ¼ 150) Root wt 0.56 24.28 12.00 4.86 Abou Kheir et al. (2008)
Root length 51.13 123.31 77.98 12.22
Root volume 6.3 100.6 50.51 19.12
TDM 38.7 394.2 215.1 19.1
R/S ratio 0.021 0.15 0.08 0.01
Note: Germplasm accessions of each of these species were raised in specially designed root structures. Plants were harvested at the grand growth stage
(flowering)
221
Maintenance of transpiration rate is crucial for productivity both under well-

watered and water-limited conditions, and hence roots play a pivotal role. Being
quantitatively inherited improvements in root traits can be best achieved with the
use of molecular marker technology. A number of studies have reported QTLs for
root architecture and have investigated their influence on yield under varying
moisture regimes in rice (MacMillan et al. 2006; Steele et al. 2006, 2007) and
Maize (Tuberosa et al. 2003; Landi et al. 2007). After identifying four major QTLs
in rice (Courtois et al. 2000), marker-assisted backcrossing was performed to
introgress the alleles for greater root length from Azucena into KalingaIII, an
upland indica variety (Steele et al. 2006, 2007). Similarly, in maize, a major QTL
originally reported for leaf ABA concentration (Tuberosa et al. 1998) was later
shown to affect root size and architecture (Giuliani et al. 2005) and grain yield
(Landi et al. 2007). These studies clearly demonstrate the possibility of enhancing
root traits, thus leading to a better field performance of crop plants under water-
limited conditions.
9.12 Transgenic Approach for Root Trait Improvement
Transgenic technology has had a great impact on crop improvement. This technol-
ogy of precision not only allows the validation of identified genes but also helps
identification of new genes. In the present scenario as well, this technology could be
exploited and utilized to check the efficacy of the identified genes and select the
gene(s) that help to obtain the right phenotype.
There are clear overexpression and downregulation studies available on the
role of specific TFs and regulatory proteins in root growth and abiotic stress
tolerance. A salt stress-inducible gene called Alfin1 (Winicov 1993; Bastola et al.
1998; Winicov and Bastola 1999), which encodes a putative Zn–finger regulatory
protein, is predominantly expressed in roots. This TF in alfalfa roots binds to
promoter elements of salt-inducible MsPRP2 gene and induces the gene expression
(Winicov and Bastola 1999). The Alfin1 from alfalfa shows conservation among
diverse plants such as rice and Arabidopsis (Bastola et al. 1998). In alfalfa, over-
expression of Alfin1 enhances root growth under normal and saline conditions,
resulting in salt tolerance (Winicov and Bastola 1999; Winicov 2000). An auxin-
induced gene called OsRAA1 was identified and characterized by reverse genetics
approach in regulating root development in rice (Ge et al. 2004). OsRAA1 is
constitutively expressed in rapidly growing cells such as primordia of the lateral
roots, meristem, and division zone of root apex. The expression of OsRAA1 is
regulated by auxin, and in transgenic rice plants, overexpressing the gene initiation
and growth of adventitious roots were more sensitive to auxin treatment (Ge et al.
2004). It has been suggested that OsRAA1 can be a candidate gene in root
development and root response to gravity. Similarly, in another study, Ca2þ-
dependent protein kinase1 (CDPK1) has been predicted to be associated with root
development in Medicago truncatula (Ivashuta et al. 2005). The TFs that regulate
diverse processes of plant development are also shown to be involved in root
growth. For example, CAP2, a gene encoding APETALA2 (AP2)-family TF, has
been shown to improve root growth and abiotic (dehydration and salt) stress
tolerance (Shukla et al. 2006). Constitutive expression of chickpea (Cicer arieti-
num) CAP2 protein in tobacco caused drastic increase in the number of lateral
roots. Overexpression of NAC1 gene enhanced lateral root formation (Xie et al.
2002). Similar to this, overexpression of Arabidopsis gene, HARDY, an AP2-
family TF, induced better root growth and imparted drought and salt tolerance
(Karaba et al. 2007).
Further, components of signaling pathways have also been identified and vali-
dated. In plants, Ca2þ is a ubiquitous secondary messenger, and changes in cyto-
solic concentration of Ca2þ are associated with plant developmental processes
including root growth. Ca2þ -dependent protein kinase 1 (CDPK1) is involved in
Ca2þ signaling events. By using RNA interference-based approach, the importance
of CDPK1 in root development was demonstrated (Ivashuta et al. 2005), and the
authors suggest that CDPK1 is a key component in signaling pathways. Similarly,
Calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK)
mediate a variety response to external stresses in plants. In Arabidopsis, CIPK6 is
required for growth and development, and tobacco plants expressing a homologous
gene (CaCIPK6) from chickpea showed improved abiotic stress tolerance (Tripathi
et al. 2009). It has been concluded that CIPK is associated with auxin transport and
consequently in root development, and salt-stress response, by regulating the
expression of downstream genes. Some of these TFs and regulatory genes can be
used to improve abiotic stress tolerance in candidate crops by transgenic approach
since some of these genes produced desirable phenotype under overexpressed
condition without abnormal phenotype.
Similarly, nutrient acquisition traits can be improved by overexpression of both
structural and functional genes involved. Overexpression of ZAT6, a zinc finger
transcription factor, resulted in altered root architecture with changes in Pi acquisi-
tion (Devaiah et al. 2007a, b).
Therefore, in a broader perspective, transgenic approach could be used to target
the modifications of the root systems with the genes involved. This could provide an
opportunity to improve the anchorage, hasten the growth of plants by enhancing
their exchange abilities, improve the tolerance of plants to drought and salinity,
their ability to penetrate compact soils, as well as synthesize important secondary
metabolites produced by the root and required by the plants.
9.13 Conclusions
Crop improvement for the future requires a very focused and orchestrated strategy
through exploitation of genomic resources. With the advent of modern molecular
biological tools, genes that regulate the growth and development of roots have been
identified. After convincing validation, several candidate genes have also been
identified that are being effectively used for improving drought tolerance through
transgenic technology. Though this technique holds tremendous potential in enhanc-

ing tolerance to severe stress levels, their field performances have not shown
significant advantage. Plants experience much milder stresses under field conditions
that are often intermittent. On the other hand, a trait improvement has had a definite
improvement in crop performance, albeit with a slower pace. To enhance the field
performance under water-limited conditions, introgressing relevant QTLs governing
root traits appears to be the most plausible strategy. Based on the stability of their
effect across environments, “constitutive” and “adaptive” QTLs have been identi-
fied. While the constitutive QTLs are consistently detected across most environ-
ments, the adaptive QTLs are detected only in specific environments. With the
advent of marker-assisted breeding technologies, it is now possible to introgress
relevant QTL for a specific target environment. However, the reliability of a QTL
entirely depends on the accurate phenotyping of the root traits in large number of
accessions and breeding lines. We have described suitable methodology for such a
large-scale screening for traits under field conditions. Phenotyping for root traits in
specially constructed root structures would be closest to that observed under field
conditions. In this approach, root traits are measured under near-natural field
condition, and hence, it is a robust phenotyping technique. Further, a more powerful
and high throughput approach based on the oxygen isotope enrichment has been
shown to be a good surrogate for root traits at a given leaf area.
Acknowledgments Most of the experiments carried out to assess genetic variability in root traits
were supported by the Department of Biotechnology under the Center of Excellence Program
support scheme and ICAR under the Niche Area of Excellence program. Graduate students Mr.
Mohan Kumar MV (Rice), Mr. Rajashekar Reddy (Finger millet), Ms. Vinoda KS (Mulberry), and
Mr. Vikram & Mr. Shashidhara (Sunflower) conducted root structure experiments. Technical help
of Dr. J.N. Madhura, Research Scientist, and Nagabhushana, Lab assistant, while analyzing
samples is sincerely acknowledged.
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Chapter 10
Genomics and Physiological Approaches
for Root Trait Breeding to Improve Drought
Tolerance in Chickpea (Cicer arietinum L.)
Rajeev K. Varshney, Lekha Pazhamala, Junichi Kashiwagi,

Pooran M. Gaur, L. Krishnamurthy, and Dave Hoisington
Contents
10.1 Chickpea Crop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
10.2 Drought Stress in Chickpea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
10.3 Strategies to Tackle Drought Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
10.3.1 Targeting Root Traits for Drought Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
10.3.2 Physiological Mechanisms of Root Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
10.4 Genetic Dissection of Root Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
10.5 Transcriptomics Approaches for Identification of Genes from Root Tissues . . . . . . . . . . 242
10.6 Prospects for Molecular Breeding for Root Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
10.7 Looking Ahead on Root Trait Research and Applications in Chickpea . . . . . . . . . . . . . . . 245
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
10.1 Chickpea Crop
Chickpea is a valuable agricultural crop of South Asia and the third most important
pulse crop in the world after dry bean (Phaseolus vulgaris L.) and field pea (Pisum
R.K. Varshney (*)

Centre of Excellence in Genomics (CEG), International Crops Research Institute for the Semi-
Arid Tropics (ICRISAT), Patancheru 502 324, A.P., India
and
Theme - Comparative and Applied Genomics, Generation Challenge Programme, c/o CIMMYT
Generation Challenge Program (GCP), c/o CIMMYT, Int APDO Postal 6641, 06600 Mexico, DF,
Mexico
and
School of Plant Biology (M084), The University of Western Australia, School of Plant Biology
(M084), Natural and Agricultural Sciences, 35 Stirling Highway, Crawley, WA 6009, Australia
e-mail: r.k.varshney@cgiar.org
L. Pazhamala, P.M. Gaur, L. Krishnamurthy, and D. Hoisington
International Crops Research Institute for the Semi-Arid Tropics ( ICRISAT), Patancheru 502324,
A.P., India
J. Kashiwagi
Hokkaido University, Graduate School of Agriculture, Kita 9 Nishi 9, Kita-ku, Sapporo 060-8589,
Japan

234 R.K. Varshney et al.
sativum L.). Cultivated chickpea, Cicer arietinum L., is a self pollinated, diploid
(2n ¼ 2x ¼ 16) annual pulse crop with a genome size of 750 Mbp (Arumuganathan
and Earle 1991). There are two types of chickpea: desi (brown colored small seed)
and kabuli (white or beige colored large seed). Desi type covers about 85% of
global chickpea area and is predominantly grown in South and East Asia, Iran,
Ethiopia, and Australia, and the kabuli type is grown mostly in the countries of the
Mediterranean regions, West Asia, North Africa, and North America. The wild
ancestor of domesticated chickpea is Cicer reticulatum. Chickpea originated in
southeastern Anatolia (Turkey) and was traditionally cultivated in Asia, the Medi-
terranean, the Middle East, and northern Africa (Ladizinsky and Adler 1976). In
contemporary times, chickpea has become popular throughout the temperate
regions in countries such as Mexico, Canada, and Australia (Duke 1981).
Chickpea ranks third among pulses, fifth among grain legumes, and 15th among
grain crops of the world. In 2006, the world chickpea cultivation area was 10.7 Mha
with over 8 Mha grown in India, Pakistan, and Iran, with a further 1 Mha grown in
other countries of Asia, the Middle East, and Canada. Total production was 8.4 Mt,
and the average yield was 772 kg/ha (FAOSTAT 2006). Although chickpea is
cultivated in about 50 countries, 95% of its area is in the developing countries
where South Asia alone covers almost 71% of the world chickpea harvested area.
Most of the chickpea harvested is consumed locally and the global trade is about
12% of the total production. The global demand for chickpea is projected to be
11.1 Mt in 2010. Under optimum growing conditions, the yield potential of
chickpea is 6 t/ha (Singh 1987), which is much higher than the current global
yield average of ~0.8 t/ha (Ahmad et al. 2005).
10.2 Drought Stress in Chickpea
The main constraints in chickpea production are the abiotic stresses such as
drought, heat, cold, and high-salinity and the biotic stresses such as Ascochyta
blight, Fusarium wilt, and the pod borer. The estimated collective yield losses due
to abiotic stresses (6.4 Mt) are higher than that of the biotic stresses (4.8 Mt) (Ryan
1997). In the order of importance, drought, cold, and salinity are the three main
abiotic stresses that affect chickpea growth and productivity worldwide (Croser
et al. 2003). Drought stress alone causes a 40–50% reduction in yield globally
(Ahmad et al. 2005). It is estimated that if the yield loss due to drought stress is
alleviated, chickpea production could be improved up to 50%, equivalent to
approximately US$ 900 million (Ryan 1997).
As 90% of chickpea crops are cultivated under rainfed conditions, drought is of
major concern (Kumar and Abbo 2001), with terminal drought as the major con-
straint limiting productivity. Terminal drought stress is typical of the post-rainy
season crop in the semiarid tropical regions, where the crop grows and matures
on a progressively receding soil moisture profile (Ludlow and Muchow 1990;
Krishnamurthy et al. 1999), and the intensity of terminal drought varies depending
10 Genomics and Physiological Approaches for Root Trait Breeding 235
on previous rainfall, atmospheric evaporative demand, and soil characteristics such

as type, depth, structure, and texture. In the arid and semiarid tropics of South and
Southeast Asia, chickpea is grown in the winter season immediately after the end of
the rainy season. Similarly in the Mediterranean environments, it is grown in spring
on stored soil moisture from the winter and early spring rainfall. In both the environ-
ments, the soil moisture recedes to deeper soil layers with the advancement in
crop growth, and the crop experiences increasing soil moisture deficit at the critical
stage of pod filling and seed development (Saxena 1984; Siddique et al. 2000).
10.3 Strategies to Tackle Drought Stress
Two main strategies are envisaged to tackle drought stress in chickpea (1) develop-
ing early maturing varieties and (2) developing drought tolerant varieties (Gaur
et al. 2008a, b). The breeding strategy for development of early maturing cultivars
is straight forward. One of the parents used in crosses should be a well-adapted
cultivar, and another parent should be an early maturity germplasm accession/
cultivar. In segregating generations, plants that flower early, for instance, in
25–30 days at ICRISAT-Patancheru, are selected and their progenies are further
evaluated. Selection for time to flower is effective even in early segregating
generations as it is controlled by a few major genes. Early flowering is a recessive
trait and controlled by a major gene ppd in ICC 5810 (Or et al. 1999) and by a major
gene efl-1 in ICCV 2 (Kumar and van Rheenen 2000). Early phenology (early
flowering, early podding, and early maturity) is the most important mechanism to
escape terminal drought stress. At ICRISAT, the chickpea breeding program has
placed high emphasis on development of early maturing varieties for enhancing
adaptation of chickpea to environments prone to terminal drought stress (Gaur et al.
2008b). Several varieties (e.g., ICCV 2, ICCC 37, JG 11, and KAK 2) have been
developed that mature in 85–100 days at Patancheru, as compared to >110 days
taken by the traditional varieties. The short-duration varieties have greatly con-
tributed to the expansion of area and enhancement of productivity of chickpea in
terminal drought-prone areas of peninsular India (Gaur et al. 2008b) and Myanmar
(Than et al. 2007). Breeding lines have been developed, which are extra-early in
maturity (75–80 days at Patancheru) and offer further opportunities for expanding
cultivation of chickpea in new niches (Kumar and Rao 1996; Gaur et al. 2008b).
Early maturing varieties that escape terminal drought and heat stress were
developed by the breeders and were adopted by farmers with considerable success
(Kumar and Abbo 2001). However, this drought escape fixes a ceiling on the
potential yield and cannot utilize the opportunities, as and when available, of
extended growing periods. Therefore, for achieving high and stable yields under
drought, it is necessary to develop drought-tolerant/avoiding varieties (Johansen
et al. 1997). Thus, several studies in the recent years have focused on identification
of morphological and physiological traits associated with drought tolerance.
Cultivated chickpea (Cicer arietinum) has a narrow genetic base, making it difficult
for breeders to produce new elite cultivars with durable tolerance to drought stress.
In addition, drought tolerance is inherited in a quantitative manner, and the direct
yield or biomass assessment under field is prone to confounded environmental
effects. Therefore, selection of drought-tolerant plants in the field becomes difficult.
Recent advances in genomics can assist crop improvement efforts (Varshney et al.
2005). In fact, marker-assisted selection (MAS) approach has been successfully
deployed in developing improved varieties/lines/hybrids in several crop species
(see Varshney et al. 2006, 2010). Quantifying the effects of drought stresses,
however, involves measurement of various factors like days to flowering and mat-
urity, early shoot growth vigor, yield, shoot biomass production, rooting depth, root
length density, root to shoot ratio, total transpiration, and transpiration efficiency.
Therefore, developing molecular markers for drought tolerance per se is a difficult
task. Dissection of such complex traits into components or identification of highly
related surrogate traits can enhance the heritability of such traits and facilitate
development of molecular markers associated with each of such traits.
10.3.1 Targeting Root Traits for Drought Tolerance
Root traits, such as root depth and root proliferation, have been identified as the
most promising traits in chickpea for terminal drought tolerance, as these help in
greater extraction of available soil moisture. As these traits are quantifiable under
drought stress conditions, it seems feasible to develop molecular markers for these
traits and thereby can be used to screen the germplasm for drought tolerance.
One of the important physiological reasons to target root traits under the water-
limiting environments is the capability of root systems to absorb relatively more
water from deeper soils and/or absorb water relatively rapidly. Chickpea is a crop
that is often grown in deeper and heavier soils such as vertisols under progressively
receding soil moisture with little precipitation during the crop growth period.
Heavier soils are characterized with soil cracking as a consequence of shrinking
when dry. These soil cracks aid in enhancing soil evaporation from deeper soil
layers, more so under increasing atmospheric evaporative demand coinciding with
the reproductive growth stage of the crop. Therefore, it becomes necessary to
maximize transpiration over evaporation (Johansen et al. 1994) and to enhance
crop growth before the water is lost in cracking heavier soils. More prolific roots at
the early stages of growth have been shown to be advantageous for such maximi-
zation as the root length density (RLD) values recorded in chickpea were subopti-
mal (Krishnamurthy et al. 1996; Kashiwagi et al. 2006). However, root prolificacy
may not be expected to maximize transpiration in environments where the evapo-
rative demands are too extreme, and also this trait may not help under environments
characterized with excessive vegetative growth and poor partitioning. Similarly,
deeper rooting or higher proportion of deeper root length can help in mining water
from deeper soil profiles, provided the soil profiles are fully saturated in the
previous rainy season or the soils are deep enough for the roots to penetrate.
Under such soil conditions, transpiration (T) gets maximized over evaporation,
which can increase the total water loss under water-limited conditions. The rela-
tionship of grain yield to water-related parameters has been described by Passioura
(1977) and Fischer (1981) as:
Yield ðYLDÞ ¼ Transpiration ðTÞ Transpiration Efficiency ðTEÞ

Harvest Index (HI):
The above formula indicates that the grain yield under drought could be
improved through improving any one or the combinations of the above compo-
nents. Also, these yield components have been shown to interact with each other.
For example, the timing of water availability is shown to affect the HI. Providing
small amounts of water across the growing period in comparison to the application
of all the water that is required at one time was shown to favor the wheat yields
through improved HI (Passioura 1977). Also, a deeper root system was found to be
associated with better HI and seed yield in chickpea (Kashiwagi et al. 2006). As
compared to HI, the other two factors, T and TE, can be improved by relatively less
efforts. The total shoot biomass can be increased either by increasing T or TE.
In some legume crops, e.g., common bean (White and Castillo 1990), ground-
nuts (Wright et al. 1991), and soybean (Cortes and Sinclair 1986), deep root
systems have already demonstrated to have positive effects on seed yield via
improved T. These studies emphasize that the T improvement strategy for better
soil moisture absorption through root systems could be applied in drought tolerance
breeding program in general or at least in legumes. However, until recently, little
breeding effort has been made to improve the root systems for seed yield or shoot
biomass under drought environments in chickpea. The reasons include the lack of
techniques that allow for large scale screening of genotypes, limited information on
genetic variability in root traits, and poor understanding of the genetics of root
attributes. It is also important to note that while targeting root traits in several crops
has been successful to tackle drought stress in several crops, the root traits may not
work in all environments.
At ICRISAT, near Patancheru in southern India (altitude: 545 m above the mean
sea level, latitude: 17 270 N, longitude: 78 280 E), a team of multidisciplinary
scientists has been working on root traits to improve the chickpea productivity.
More than 1,500 chickpea germplasm accessions plus released varieties were
evaluated under rainfed as well as irrigated field conditions at ICRISAT to gather
information on the yield under terminal drought conditions and potential yields
(Saxena 1987, 2003). Some genotypes, e.g., Annigeri, ICC 4958, ICC 10448, ICC
5680, and JG 62, were identified as drought-tolerant lines using a drought-tolerant
index in which the effects of early flowering could be removed (Saxena 1987),
although each had a different trait/mechanism to cope with the terminal drought.
For example, in Annigeri and ICC 10448, narrow (lanceolate) leaves, in ICC 5680
fewer pinnules per leaf and a rapid rate of grain filling through production of twin
pods at the early flowering nodes in JG 62 seem to be the mechanism contributing to
drought tolerance. The genotype, ICC 4958, showed the best performance not only
at ICRISAT field trials but also at several other locations in India and in the
Mediterranean climate in Syria, which was found to possess higher root biomass
(ICARDA 1989; Saxena et al. 1993; Krishnamurthy et al. 1996; Ali et al. 1999,
2005). Subsequently, field experiments at ICRISAT with 12 diverse chickpea
germplasm including ICC 4958 showed that a prolific root system, especially in
the 15–30 cm soil depth, had positive effects on seed yield under moderate terminal
drought intensity, and a deeper root system to improved yield under severe terminal
drought conditions (Kashiwagi et al. 2006). The large variation in root systems
within such a small group of genotypes (Fig. 10.1), and the relation between root
length density (RLD) and yield under drought, suggests that an extensive and
systematic screening of the chickpea germplasm might offer a promising range of
variation for RLD. Furthermore, the RLD was increased under more severe stress
conditions, particularly in more tolerant genotypes, and the RLD at the deeper layer
was related to yield under more severe drought stress. These data suggest that the
dynamics of root growth under drought conditions might be a key factor in
understanding the contribution of roots to drought tolerance.
Fig. 10.1 Comparative root profiles in three chickpea genotypes. The figure shows 35-day-old
plants of three chickpea genotypes, namely ICC 4958, KAK 2, and Annigeri. These plants were
grown in pots in glasshouse conditions. It is evident from the figure that the root biomass for ICC
4958 is relatively higher than the other two chickpea genotypes. Higher root biomass confers high
level of drought tolerance in ICC 4958 genotype.
The research on root systems under field conditions is very laborious, expensive,
and time-consuming (Subbarao et al. 1995). To overcome this problem, a modified
monolith method was standardized at ICRISAT (Serraj et al. 2004). This method
provided systematic field root extraction at a sampling rate of 3.3 root profiles/
worker/day. Although this method was fairly reliable to assess the field perfor-
mance, it still did not provide an adequate sampling rate for large scale screening of
genotypes. Although the less cumbersome pot-culture method was tested, the
rooting profile could not be estimated in shallow pot grown plants. Thus, extensive
efforts were made at ICRISAT to standardize a PVC cylinder-culture system for
screening large numbers of genotypes. When the plants were grown in PVC
cylinders (18 cm diameter, 120 cm height) filled with a sand–vertisol mixture
containing a 70% field capacity soil moisture, the extracted root biomass was
significantly correlated with the ones extracted from the field (r ¼ 0.62,
p < 0.05) (Kashiwagi et al. 2006). Moreover, the sampling efficiency of chickpea
roots could be improved upto 25 profiles/worker/day. Furthermore, an image
capturing and analysis system was introduced to scan the roots and convert
the intact root samples into digitalized images for a large number of samples
(>150 root samples/day). By using the digital image of roots, the WINRHIZO
software (Regent Instruments, Inc., Canada) could generate numerical data, e.g.,
root length and root diameter, from more than 500 images/day.
10.3.2 Physiological Mechanisms of Root Traits
Plants take up water from soil profile using either an active or a passive water
uptake pathway (Hirasawa et al. 1997). In nonstress conditions, i.e., when a plant
transpires, the magnitude of active water uptake is far less than that of passive water
uptake. Under severe drought conditions, however, the plants close the stomata,
so as not to deplete the internal water, and active water uptake becomes more
important under such non-transpiration situations. In active water uptake, one of the
relevant root-related traits would be osmotic adjustment. However, using such traits
is difficult in breeding programs (Turner et al. 2006).
The passive water uptake takes place by gradient of water potential from the
roots to shoots, where Vapor Pressure Deficit (VPD) in the air is the principle
driving force. Thus, higher VPD causes more transpiration to occur via stomata,
which pulls down the leaf water potential. Subsequently, it reduces the xylem
pressure potential in the stems and then in the roots. This creates a gradient in
water potential, which forces the soil water into the xylem in roots and then to the
leaves. Under normal circumstances, this passive water uptake plays a major role in
terms of the plant water. Under the passive water uptake, the relevant root traits
are root hydraulic conductivity (vertical water flow from roots to leaves) and root
permeability (transverse water flow from the root surface to xylem). The root
permeability could be further dissected into three different paths (1) apoplastic
(inter-cells), (2) symplastic (cell-to-cell), and (3) transcellular (cell-to-cell) (Steudle

2000). The symplastic path more closely relates to the active water uptake.
Chickpea is known to have varying root distribution across soil depths depend-
ing on the soil water availability. It has substantially smaller RLD than that of
several cereals, e.g., barley (Thomas et al. 1995), but has an efficient water uptake.
The difference for water uptake between chickpea and cereal species has been
attributed to the function of root hydraulic conductivity, which is mainly governed
by the diameter and the distribution of the meta-xylem vessels (Hamblin and
Tennant 1987). Chickpea could develop its root systems upto two to three times
greater in the surface soil layer (0–15 cm) at mid-pod filling stage when irrigated.
On the other hand, the proportion of RLD distributed at deeper soil layers
(115–120 cm) was found higher under receding soil water conditions compared
to that of the well-watered condition (Ali et al. 2002). In another study, chickpea
had a greater proportion of the root system in the deeper soil layer under dryland
environments than field pea (Benjamin and Nielsen 2006). In addition, chickpea
possesses greater root surface area to root weight ratio, compared to field pea or
soybean. These studies suggest that chickpea plants are better equipped in terms of
the soil water uptake to cope with the drought environments. Enhancing root traits
would, therefore, be one of the promising approaches to improve drought avoidance
in chickpea under terminal drought conditions.
10.4 Genetic Dissection of Root Traits
In order to target the root traits in chickpea breeding to improve drought tolerance,
understanding the genetics of root traits is crucial. In the first instance, to have a
knowledge about the genetic variability of root traits in chickpea germplasm, a mini
core collection consisting of 211 chickpea genotypes developed by Upadhyaya and
Ortiz (2001) was assessed in the cylinder culture with image capturing and analysis
systems in two seasons. A large and significant variation was observed among the
accessions of the mini-core collection in terms of root length density (RLD), root
dry weight (RDW), rooting depth (RDp), and root to total plant weight ratio (R/T)
(Krishnamurthy et al. 2004; Kashiwagi et al. 2005). Although a significant geno-
type season interaction was observed for RLD and R/T, it was a noncrossover
type. Therefore, a rank correlation analysis was performed between the accession
means of two seasons to identify the contrasting genotypes in terms of root traits.
The studies identified two accessions namely, ICC 4958 and ICC 8261, as having
large and prolific root systems. In addition, the root traits of ten accessions of annual
wild Cicer species were also evaluated in one season. The wild relatives had smaller
root systems than C. arietinum except for the most closely related species
C. reticulatum whose root systems were similar to that of the average root system
of C. arietinum. It has to be mentioned here that these findings need further
validation keeping in mind the effect of phenology on the timing of root growth.
Most of the wild accessions tested here were late in flowering, and these evaluations
have been carried out using 35-day-old plants. As most of the wild Cicer species are
late in phenology, it may be appropriate to measure the root system differences of
wild species accessions at a later growth period.
Subsequently, in a study conducted to estimate the gene effects for root traits,
two contrasting pairs of chickpea genotypes, ICC 283 and ICC 1882 (smaller roots)
and ICC 8261 and ICC 4958 (larger roots), were identified for developing popula-
tions for the genetic analysis (Kashiwagi et al. 2008). In these analyses, the additive
gene effect and additive additive gene interaction have been found to play
important roles in determining the RLD and RDW. In addition, the direction of
the additive gene effects was consistent and toward increasing the root growth. The
results encouraged the ICRISAT team to proceed with the breeding program for
root systems in chickpea, although delaying selections until later generations with
larger populations was proposed (Kashiwagi et al. 2008).
In order to identify the genomic regions or quantitative trait loci (QTLs) for root
traits, three recombinant inbred line (RIL) populations were developed at ICRI-
SAT. The first population consists of 257 RILs from the cross Annigeri ICC
4958. Two other RIL populations involving parents more genetically and pheno-
typically distant, selected after screening the mini core collection as mentioned
above, were developed: 281 RILs from the cross ICC 283 ICC 8261 and 264
RILs from the cross ICC 4958 ICC 1882.
The Annigeri ICC 4958 RILs were evaluated for two seasons under termi-
nal drought conditions, and approximately 40 molecular markers (SSR) were
genotyped in the population. A QTL responsible for 33% of the phenotypic
variation for root length and root biomass was detected (Chandra et al. 2004).
The root trait phenotyping has been done for the two other mapping populations
(ICC 4958 ICC 1882 and ICC 283 ICC 8261), and genotyping is underway
with a variety of molecular markers. Limited level of polymorphism in intra-
specific mapping populations of chickpea is a major constraint in mapping of
any trait in chickpea. To aid in mapping, a set of 311 SSR markers have been
developed from an SSR-enriched genomic DNA library (Varshney et al. 2007),
and a set of 1,344 SSR markers have been developed after mining about 46,270
BAC-end sequences (Nayak et al. 2008). With the existing set of SSR markers in
public domain and newly developed markers at ICRISAT (in collaboration with
University of California, Davis, CA, USA; University of Frankfurt, Germany)
and National Institute of Plant Genome Research (NIPGR), New Delhi, India
(Sabhyata Bhatia, pers. commun.), more than 2,000 SSR markers are available in
chickpea (Varshney et al. 2008, 2009a; Nayak et al. 2010). An integrated genetic
map with 521 loci has been developed by Nayak et al. (2010). In addition to
SSR markers, Diversity Arrays Technology (DArT) markers are currently being
used for genotyping the two mapping populations (ICC 4958 ICC 1882 and ICC
283 ICC 8261). Given the large phenotypic and genotypic contrast between
the parents involved in these populations and high density marker genotyping,
the chances to identify additional major QTLs for root traits as defined above
are high.
10.5 Transcriptomics Approaches for Identification of Genes

from Root Tissues
Plant stress responses are complex and diverse, and every gene involved, from
recognition to signaling to direct involvement, forms part of a coordinated response
network. Controlling gene expression is one of the key regulatory mechanisms used
by living cells to sustain and execute their functions. Although the final activity of a
gene is determined by encoded protein, measurements of mRNA levels have proven
to be a valuable molecular tool. In order to obtain a complete picture of a plant’s
response to stress, it would be ideal to study the expression profiles of all possible
genes in its genome or at least those involved in conferring stress tolerance.
Traditional approaches for undertaking genome-wide expression studies involve
the use of microarray or cDNA macroarrays. Although in chickpea, transcriptomic
approaches are not in an advanced stage, they progress in this direction that has
already been initiated (Coram and Pang 2007).
The first step toward transcriptomics studies is the identification or cataloging
of genes involved in the trait. One of the most simple and straight forward approach
is the generation of expressed sequence tags (ESTs), which involves large-scale
single-pass sequencing of randomly selected clones from cDNA libraries con-
structed from mRNA isolated at a particular developmental stage and in response
to a particular stress (Sreenivasulu et al. 2002). Functional identification of sequenced
clones is becoming easier by the availability of rapidly growing sequence data-
bases, such as Genbank and genome sequence data of several crop species including
the three legumes, i.e., Medicago truncatula, Lotus japonicus, and Glycine max.
The EST datasets can be used in gene expression/functional genomics studies to
identify putative genes with differential expression and to generate the gene-based
functional molecular markers such as EST-SSRs, EST-SNPs, and single feature
polymorphisms (SFPs) (Varshney et al. 2005). EST analysis has become a popular
method for gene discovery and mapping in cereal crops (Varshney et al. 2006). The
first resource of ESTs (ca. 2800) in chickpea was developed at ICRISAT from root
tissues challenged by drought stress (Buhariwalla et al. 2005; Jayashree et al. 2005).
The EST library was constructed after subtractive suppressive hybridization (SSH)
of root tissue from two chickpea genotypes (the landrace ICC 4958 and a popular
local variety Annigeri), which were considered to possess important sources of
drought tolerance (Saxena et al. 1993; Saxena 2003). A total of 2,179 ESTs were
generated with putative identification that resulted into 477 unigenes. A total of 106
EST-based markers were designed from the unigene sequences with functional
annotations. To enrich the resource of ESTs involved in drought and salinity stress
tolerance (or response), ten different cDNA libraries were constructed from the root
tissues of ICC 4958, ICC 1882, JG 11, and ICCV 2 (parental genotypes of the
mapping populations segregating for drought and salinity), challenged by different
types of drought (chemical induction using polyethylene glycol (PEG), sudden
dehydration stress, slow drought stress to potted plants grown in the greenhouse,
and prolonged drought stress under field conditions) and salinity stresses (treated
with 80 mM NaCl solution). In summary, a total of 20,162 ESTs have been

generated in the study using Sanger sequencing approach at ICRISAT and have
been deposited in GenBank (Varshney et al. 2009b). A detailed analysis of ESTs
has provided a set of 6,404 unigenes.
In addition, “whole transcriptome sequencing” using Solexa sequencing tech-
nology (see Varshney et al. 2009c) has been initiated by ICRISAT in collaboration
with colleagues from the National Center for Genome Resources, Santa Fe, New
Mexico, USA (Greg May and Andrew Farmer), and the University of California,
Davis, USA (Doug Cook). In this approach, the RNA isolated from drought stress
challenged root tissues of different stages and were pooled for ICC 4958 and ICC
1882 genotypes separately. Half run of Solexa sequencing on the pooled RNA
samples from ICC 4958 and ICC 1882 yielded 5.2 106 and 3.6 106 sequence
reads (May et al. 2008), respectively. The preliminary results of the Solexa
sequencing are summarized in Table 10.1. Ideally for analyzing the Solexa datasets,
genome assembly (reference assembly) of the same species is prerequisite for
aligning the short tags (~36 bp). In case of chickpea, however, no genome assembly
was available during the analysis. To analyze the generated Solexa datasets, the
following three set of sequence resources were used (1) M. truncatula (Mt) IMGAG
(International Medicago Genome Annotation Group) gene assembly representing
29.5 Mb sequence data, (2) C. arietinum transcript assembly (Ca TA) of JCVI
(The James Craig Ventor Institute) representing 681 kb sequence data and (3)
C. arietinum (Ca) BAC-end sequence (Ca BES) data representing 16.4 Mb
sequence data. As a result, the Solexa datasets showed matches with 5,886 and
7,338 genes in cases of ICC 4958 and ICC 1882, respectively (Table 10.1). These
datasets are being analyzed for identification of gene-based SNPs between ICC
4958 and ICC 1882 so that the polymorphic genes could be integrated in the genetic
maps. Such efforts should lead to the identification of drought QTL-associated
genes that would be useful for molecular breeding.
Other functional genomics studies using the chickpea/legume-based gene
microarrays have also been undertaken for identification of genes for drought
tolerance; however, these were not exclusively focused on root traits. For example,
Table 10.1 Preliminarily gene discovery in two chickpea genotypes by employing the Solexa
sequencing technology
Features ICC 4958 ICC 1882
Number of reads 36,15,433 52,07,099
Average read length 36 36
Average read quality 26 21
Alignment with TA
Read aligned 11,95,622 (33%) 21,22,069 (41%)
Reads uniquely aligned 5,72,751 (16%) 9,67,102 (19%)
Alignments with BES
Aligned 10,48,614 (16%) 17,88,936 (34%)
Uniquely aligned 5,11,148 (14%) 8,54,085 (16%)
Overall number of gene matches 5,886 7,338
Boominathan et al. (2004) carried out a gene expression study of drought adaptation
in chickpea using subtractive suppressive hybridization in combination with differ-
ential DNA array hybridization and northern blot analysis and identified 101
drought-inducible transcripts. Similarly, Coram and Pang (2006) developed a
“Pulse Chip” microarray and applied it to identify the genes expressed in response
to abiotic stresses such as drought, cold, and high salinity. In another study,
transcript profiling of tolerant and susceptible chickpea genotypes under drought,
cold, and high salinity was conducted (Mantri et al. 2007). These studies provide
opportunities for illuminating the mechanisms of drought tolerance in chickpea and
indicate the molecular pathways used by the plant as well as the function of the
candidate genes involved. It would be interesting to see the colocalization of such
genes with QTLs related to root trait in chickpea.
10.6 Prospects for Molecular Breeding for Root Traits
The role of root traits in conferring drought tolerance in chickpea is well estab-
lished. A significant challenge to the selection for root traits is the difficulty of
evaluating root phenotypes, since many root traits are phenotypically plastic, roots
are difficult to extract from the soil, such extraction may change certain traits such
as architecture, and many root sampling procedures are destructive. Research on
drought tolerance still has to deal with many complicated aspects, especially
concerning root functions. The reason is that the root is difficult to visualize and
extremely sensitive to the surrounding environmental factors because of the G E
interactions. So, many efforts have been made to characterize and identify varietal
differences based on root traits (Kashiwagi et al. 2005). These challenges make
the prospects of marker-aided selection an attractive alternative to phenotypic
selection.
The availability of appropriate molecular markers is an important prerequisite
for marker-assisted selection. The availability of more than 2,000 SSR markers
and DArT arrays in chickpea will enable the development of the genetic maps
and mapping of traits in intraspecific populations. The integration of the candid-
ate genes showing differential expression as well as SNPs between contrasting
genotypes into QTL maps will provide genes and markers associated with root
trait QTLs.
After identifying the QTLs, molecular markers associated with these QTLs
need to be validated on a range of germplasm to select the most promising QTLs.
For introgression of these QTLs, the drought-tolerant (possessing the QTLs) and
drought-sensitive lines (showing the polymorphism at QTL with drought tolerant
genotypes) are selected. After generating the F1s by crossing the susceptible
drought-sensitive varieties (recurrent) with drought-tolerant donor variety, the F1
seeds are raised and backcrossed to the recipient varieties. After raising the BC1F1
population, these plants are genotyped with the identified molecular marker(s)
associated with targeted QTLs. Based on marker genotyping data, the desired plants
are used further for backcrossing to produce the BC2F1 populations. Similar cycles
of backcrossing and selection of lines with molecular markers for making them
homozygous for the next generations are continued until the necessary recovery of
the recurrent parent genotype is achieved. Many molecular breeding programs do
not involve the use of markers in background selection. However, the availability of
Diversity Array Technologies (DArTs), a low cost marker system in chickpea,
creates the possibility to use DArT markers for background selection. Subse-
quently, the marker-assisted backcross (MABC) lines are evaluated in replications
on-station and on-farm trials for agronomic performance. Eventually, the successful
products of MABCs are selected and advanced to release as varieties in targeted
environments.
Indeed, the above scheme of introgressing of QTLs/genes into varieties of
interest has been successfully utilized in several cereal species (Varshney et al.
2006, 2007). It is anticipated that introgression of root trait QTLs in drought-
sensitive chickpea varieties should be feasible in the coming years.
10.7 Looking Ahead on Root Trait Research and Applications

in Chickpea
This chapter presents the importance of root traits in conferring drought tolerance in
chickpea. However, molecular mechanisms of root traits at the physiological and
genetic level are yet to be understood. On the one hand, the simple screening
methods have been developed for precise phenotyping root traits at a large scale,
enabling phenotyping of large segregating populations possible. In parallel, the
genomic resources including large number of SSR markers, BAC and BIBAC
libraries, BAC-end sequences, ESTs, and Solexa tags have been developed (Varshney
et al. 2009a). These resources offer the possibility to develop the dense genetic
map, transcript maps, and integrated genetic-physical maps of chickpea. These
genomic tools should identify the root trait QTLs at a higher resolution that can
be used in molecular breeding for drought tolerance in chickpea.
In order to understand the genetic basis of root traits at the molecular and cellular
level, it will be possible to delimit root trait QTLs and dissect them at nucleotide
level with the help of genomic resources in chickpea as well as in M. truncatula,
L. japonicus, and G. max by using comparative genomics. The approaches like
“genetical genomics” or “expression genetics” that involves the analysis of gene
expression data together with the phenotyping data should provide the insights on
direct involvement or regulation of QTL/gene for root trait on drought tolerance.
The function of candidate genes can further be validated by using the chickpea
TILLING populations recently developed at Washington State University, USA
(Rajesh et al. 2007), and ICRISAT. With such available resources, we envision a
more rapid understanding of the genetic and functional basis of root traits for
drought tolerance.
Phenotyping Chickpea Molecular

root traits germplasm marker analysis
Mapping Reference
populations collection
Genome-wide
Phenotyping marker analysis
Candidate gene
sequencing
Genes, Marker
and QTLs
Introgression through
marker assisted breeding
Improved
germplasm
Fig 10.2 A scheme to utilize the root traits for chickpea improvement. The figure represents the
holistic approach combining genomics, physiological, and breeding strategies. For instance, the
molecular marker profiling and physiological screening of germplasm provides the contrasting
genotypes at genetic as well as physiological level for developing (a) the mapping populations and
(b) the reference collection. The mapping populations can be genotyped with molecular markers
and phenotyped for root traits. Linkage analysis together with phenotyping data on the mapping
population will provide the QTLs and markers associated with root traits. Similarly, the genome
wide molecular genotyping or candidate gene sequencing of the reference collection together with
phenotyping data for root traits can be subjected for association genetics and the markers/genes
tightly associated with root traits can be identified. Molecular markers/genes identified by linkage
analysis or association genetics can be used for marker-assisted breeding to introgress the drought-
tolerant genomic regions from drought-tolerant genotypes into drought-sensitive genotypes to
develop improved drought-tolerant cultivars of chickpea
Finally, the advancement in chickpea genomics and refinement of root physiol-

ogy approaches would provide access to agronomically desirable alleles present at
QTLs for root traits. A scheme has been proposed in Fig. 10.2, showing the
utilization of root traits for chickpea improvement. The combined approach of
genomics and physiology in chickpea breeding would enable us to improve the
drought tolerance and yield of chickpea under water-limited conditions more
effectively.
Acknowledgments Authors are thankful to colleagues involved in root trait research in chickpea
at ICRISAT for sharing the published as well as unpublished results. Thanks are due to Generation
Challenge Program (http://www.generationcp.org), National Fund of Indian Council of Agricul-
tural Research (ICAR), and the Department of Biotechnology of Government of India for
sponsoring the research projects to carry out the research on drought tolerance and chickpea
genomics.
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Chapter 11
Molecular Breeding of Cereals for Aluminum
Resistance
Harsh Raman and Perry Gustafson
Contents
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
11.2 Evaluation of Germplasm for Aluminum Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
11.3 Genetic Variability for Al Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
11.4 Genetic Control of Al Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
11.5 Molecular Mapping of Al Resistance Loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
11.5.1 Rye and Triticale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
11.5.2 Barley . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
11.5.3 Oat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
11.5.4 Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
11.5.5 Maize and Sorghum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
11.6 Molecular Synteny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
11.7 Mechanism of Aluminum Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
11.8 Functional Genomic Approaches in Elucidating and
Validating Al Resistance Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
11.8.1 TaALMT1 Gene Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
11.8.2 Homologs and Paralogs of TaALMT1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
11.8.3 MATE Gene Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
11.8.4 Expression Analysis of MATE and ALMT1 Homologs . . . . . . . . . . . . . . . . . . . . . 269
11.9 Discovery of Candidate Genes Expressed Under Al Stress . . . . . . . . . . . . . . . . . . . . . . . . . . 271
11.10 Molecular Breeding for Al Resistance Using Genetic Transformation . . . . . . . . . . . . . . 272
11.11 Molecular Breeding for Al Resistance Using Marker-Assisted Selection . . . . . . . . . . . . 273
11.12 Allele Mining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
11.13 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
H. Raman
NSW Department of Industry and Investment, Wagga Wagga Agricultural Institute, Wagga
Wagga, NSW 2650, Australia
e-mail: harsh.raman@dpi.nsw.gov.au
P. Gustafson (*)
USDA-ARS, University of Missouri, 206 Curtis Hall, Columbia, MO 65211, USA
e-mail: Perry.Gustafson@ars.usda.gov

252 H. Raman and P. Gustafson
11.1 Introduction
Soil acidity limits the production of cereals on over 1.5 billion hectares worldwide
and possesses a serious threat to world food security (FAO stat). Crop productivity
on acid soils is often restricted due to multiple stresses. On acid soils, there are
several limiting factors for plant growth, including toxic levels of aluminum (Al3+),
manganese, and iron, as well as deficiencies of essential elements, such as phos-
phorus, nitrogen, potassium, calcium, magnesium, and some micronutrients (2004).
Al toxicity is a major factor limiting crop production on highly weathered acid soils
(Kochian 1995). The Food and Agriculture Organization of the United Nations
(FAO) lists Al toxicity as affecting 14% of all soils worldwide, with the level
greater than 50% in many countries (http://www.fao.org/ag/agl/agll/terrastat/wsr.
asp#terrastatdb). At low pH (<5), dissolution of Al-containing compounds is
enhanced and the release of toxic Al3+ cations into soil solution can rapidly inhibit
root growth (Delhaize et al. 1993b). Subsequently, Al toxicity may inhibit supply of
nutrients, growth hormones, and water mainly due to poorer root penetration (Pan
et al. 1989).
A number of key cereal crops such as wheat (Triticum ssp.) (Polle et al. 1978),
rice (Oryza sativa L.) (Nguyen et al. 2001), maize (Zea mays L.) (Ceretta 1988;
Mazzocato et al. 2002), barley (Hordeum vulgare L.) (Reid et al. 1969), sorghum
(Sorghum bicolor L.) (Blamey et al. 1992), and rye (Secale cereale L.) (Gallego and
Benito 1997) are sensitive to Al toxicity. The Al resistance order has been reported
as maize > rye > triticale (X Triticosecale Wittmack) > wheat > barley (Polle and
Konzak 1985), rye > oat (Avena sativa L.) > millet (Panicum miliaceum L.) >
bread wheat (T. aestivum L.) > barley > durum wheat (T. turgidum ssp durum L.)
(Bona et al. 1993), and rice > maize > pea > barley (Ishikawa et al. 2000). Most
Al-sensitive genotypes show greatly reduced root growth and either die within a
few weeks after germination on acidic soils or yield very poorly. The effects of Al
toxicity can be more pronounced under drought and heat stress environments.
Symptoms of Al phytotoxicity are not always easily identified in the field;
however, the initial and the most dramatic symptom of Al toxicity is inhibition of
root elongation, which can occur within minutes of exposure to micromolar toxic
concentrations of Al3+. Aluminum permeates the plasma membrane and accumu-
lates in the root tips (Samuels et al. 1997). The root apex, where cell division and
elongation occurs, is recognized as the main site of Al accumulation and toxicity in
sensitive cultivars (Delhaize et al. 1993b; Sivaguru and Horst 1998). However, in
maize, distal transition zone is the most Al-sensitive region in the Al-sensitive
cultivars such as “Lixis” (Kollmeier et al. 2001; Sivaguru and Horst 1998). Alumi-
num results from the binding of Al to extracellular and intracellular substances
because of the high affinity of Al for oxygen donor compounds. When the root
elongation is inhibited by Al, most of the Al is localized on the epidermis and the
outer cortex (Jones et al. 2006).
Two strategies have been used to extend cultivation and enhance yield per unit
area on acid soils (1) an application of lime for neutralizing the acidity and/or (2)
11 Molecular Breeding of Cereals for Aluminum Resistance 253
cultivation of Al-resistant varieties. An application of lime is often not practical

because of its slow movement, especially in the deeper layers of subsoils (Foy et al.
1965; Mugwira et al. 1976), and adequate liming may not be economically feasible
in many regions of the world (Pandey et al. 1994), especially in low-yielding
environments. In addition, heavy application of lime may also have adverse effects
on some crops in the rotation or cause deficiencies of certain nutrients (Rao et al.
1993; Whitten 1997). In the literature, both the terms Al tolerance and Al resistance
have been used interchangeably. In this review, the term Al resistance is used to cite
relevant research on Al resistance/tolerance in cereals.
There are two prerequisites for exploiting resistance genes in breeding programs
to develop new varieties (1) presence of genetic variability for Al resistance and (2)
understanding the genetic control of Al resistance within the species involved.
Genetic variability for Al resistance has been reported among the cultivated and
wild germplasm of wheat, barley, rice, rye, oats, sorghum, and maize (Ali et al.
2008; Cançado et al. 1999; Ceretta 1988; Minella and Sorrells 1992; Raman et al.
2008a, b, c; Read and Oram 1995; Reid et al. 1969; Silva et al. 2006) and has been
exploited in breeding programs to develop high-yielding varieties with greater
resistance to Al toxicity. Crop improvement programs worldwide have developed
hundreds of varieties suitable for cultivation on acid soils.
In this chapter, we will describe new advances in understanding of genes and
gene complexes conditioning Al resistance in cereals and their further use in
molecular breeding via marker-assisted selection and genetic engineering.
11.2 Evaluation of Germplasm for Aluminum Resistance
Most methods for testing Al resistance in plants are based upon inhibition of root
growth due to Al toxicity. Different methods have been employed for screening
germplasm for aluminum resistance including nutrient solution culture (hydropon-
ics), pot assays in the glasshouse, and field evaluation on acidic soils; see review
Wang et al. (2006a). However, laboratory and greenhouse-based techniques are
widely employed, which are usually nondestructive, and can be performed in early
stages of plant development from, only a few days-old seedlings to flowering stage
of the plants. There are several advantages of the nutrient solution method over the
soil-based assays. In nutrient solution culture, the dose of Al3+ and conditions (e.g.,
pH, light, temperature, humidity, etc.) for screening plants can be precisely con-
trolled. Root measurements from the nutrient solution culture method are much
easier as compared with soil assays, as the primary effect of Al toxicity on the plants
is the inhibition of root elongation, and the roots are easily observed under nutrient
culture. However, root growth measurements are relatively more time-consuming.
Root measurements are also dependent upon concentration of particular ions
(nutrient status of solution), genetic vigor, and age of seedlings.
Nutrient solution culture-based evaluation is more suited for large-scale screen-
ing of germplasm for Al resistance. Several hundred seedlings can be evaluated for
Al resistance, within a week, in a small space, whereas soil-based assays are more
labor-intensive, expensive, and require additional glasshouse space. Under nutrient
solution culture, Al resistance has been evaluated using hematoxylin (Cançado
et al. 1999; Polle et al. 1978), eriochrome cyanine staining (Furukawa et al. 2007;
Gruber et al. 2006; Magalhaes et al. 2004), root growth (Raman et al. 2005), and
relative root regrowth (Raman et al. 2002, 2005, 2008c). Hematoxylin and erio-
chrome cyanine stain-based methods are based on the ability of Al-resistant seed-
lings to continue root growth following a short pulse treatment involving a high Al
concentration, while the relative root growth (RRG) method uses the root growth
and root resistance index to judge Al resistance over a period of time (usually 2–4
days). Root elongation has been suggested to be one of the most important markers
when screening genotypes and cultivars for Al toxicity (Taylor and Foy 1985).
Since root growth under Al stress is a combination of root vigor (long roots) and Al
resistance, selection of Al resistant genotypes using RRG is preferred as it allows
for a better differentiation of genotypes, and it is often used to measure relative
level of Al resistance. For example, Hede et al. (2002) evaluated 63 rye accessions
from a world spring rye collection for Al resistance using the hematoxylin and the
root growth method and demonstrated that the hematoxylin method and the root
growth parameter identified genotypes with long root growth under Al stress, but it
failed to detect Al resistance in genotypes with poor root vigor. RRG/relative root
length or root resistance index has been measured as
RRG ¼ Root growth under Al stress=control root length ðve AlÞ 100:
This technique is very simple to measure and eliminates the genetic difference in
root vigor under nutrient culture. Massot et al. (1992) showed that scoring for Al
resistance, using root elongation as a single criterion, may avoid the misclassifica-
tion of genes, which allows for the accumulation of a large amount of Al in shoots.
Nutrient culture-based selection for Al resistance has been highly correlated with
hematoxylin stain method and field evaluation. Stodart et al. (2007) compared the
minimum and maximum root regrowth measures and reported a good relationship
between hematoxylin score and the regrowth measure (Fig. 11.1).
Baier et al. (1995) suggested that hydroponic screening of wheat seedlings for Al
resistance may be used in breeding programs or in screening germplasm collections.
This study correlated root lengths of 43 wheat genotypes grown in Al-containing,
acidic hydroponic solutions with root weights from acid-soil experiments and field
scores from Brazilian acid-field trials and revealed highly significant correlations
(r ¼ 0.71–0.85) between root length or a root tolerance index in the Al solutions vs.
acid-soil experiments and acid-field trials.
Besides the hematoxylin and eriochrome staining methods, Maltais and Houde
(2002) reported that nitroblue tetrazolium (NBT) reduction is a simple biochemical
marker that is correlated with the degree of Al resistance in wheat, rye, maize, and
rice. All the plants were able to grow, demonstrating that this scoring technique is
rapid and nondestructive. This Al resistance marker was the first to provide a strong
signal in resistant plants rather than in sensitive plants (Bennet 1997; Horst et al. 1997;
14 Partial Staining Stained Unstained
Carrazhino
12
10
minimum re-growth (mm)
0
0 2 4 6 8 10 12 14 16 18 20
maximum re-growth (mm)
Fig. 11.1 Comparison of 250 wheat landrace accessions for Al tolerance evaluated using root
regrowth measurements and hematoxylin staining (Unstained: Al-resistant, Stained: Al-sensitive,
and Partial stained: Intermediate Al-resistant). Al-tolerant reference cultivar, Carrazhino is
indicated (after Stodart et al. 2007)
Massot et al. 1992, 1999). NBT test is suitable for screening thousands of plants in a
single day/person (Maltais and Houde 2002).
11.3 Genetic Variability for Al Resistance
Natural genetic variability for resistance to Al exists within different species of

cereals (Bernal and Clark 1997; Bona et al. 1993; Khan and McNeilly 1998;
Khatiwada et al. 1996; Pineros et al. 2008; Sivaguru et al. 1992). Among cereal
crops, rye is the most resistant cereal (Aniol and Gustafson 1984; Aniol and Madej
1996; Hede et al. 2002; Little 1988; Mugwira et al. 1976), whereas durum wheat is
the most Al-sensitive (Bona et al. 1993). In the literature, very limited genetic
variability for aluminum resistance has been reported in tetraploid wheats. Raman
et al. (2008a) evaluated 408 genotypes of the subspecies durum, dicoccon, and
carthlicum of Tritticum turgidum (2n ¼ 4x ¼ 28, genomes¼AABB) for Al resis-
tance using nutrient solution culture techniques. The authors used a new measure
“Incremental crop tolerance (ICT)” that reflect the incremental root regrowth
between genotypes associated with Al resistance, over and above difference in
underlying root vigor. Statistical analysis indicated that three accessions were
Al-resistant in a nutrient solution containing 20 mM of Al. The genetic identity

(AABB) of these genotypes was confirmed using genome-specific markers Dgas44,
TaALMT1, QSSR (domestication gene-based marker), and gamma gliadin. Despite
extensive use of interspecific and intergeneric hybridization to introgress genes for
Al resistance, only few wild species have been utilized. Berzonsky and Kimber
(1986) evaluated various accessions of goat-grass Aegilops uniaristata (2n ¼ 2x
¼ 14, genome: NN) and identified useful variation for Al resistance and exploited
further to improve Al resistance in wheat (Iqbal et al. 2000).
Rye has been used to introgress superior alleles into wheat, without much
success. Triticale, a wheat/rye hybrid, is recognized as particularly Al resistant
cereals and is adequate for cultivation in acid soils (Antunes et al. 1996). Among the
triticales evaluated, “Arabian” ranked higher in resistance with only 30% reduction
in the root growth in contrast with “Beagle,” which presented the strongest inhibi-
tion (75%). Zhang et al. (1999) performed comparative analyses of genetic varia-
bility of Al resistance response in a range of triticale genotypes consisting of six
Australian cultivars, eight South African lines, and an Australian control utilizing
a solution culture technique and screening under controlled growth cabinet con-
ditions. Results showed that “Tahara,” Tahara “S,” and “Abacus” were the most
Al-resistant triticales among the Australian genotypes in terms of root regrowth
characteristics at 10 mg/g Al.
11.4 Genetic Control of Al Resistance
The genetics of Al resistance in cereals is reasonably well-understood. Monogenic

inheritance for Al resistance has been reported in various populations of wheat
(Baier et al. 1995; Delhaize et al. 1993b; Johnson et al. 1997; Kerridge and
Kronstad 1968; Luo and Dvorak 1996; Milla and Gustafson 2001; Raman et al.
2005; Somers et al. 1996); barley (Ma et al. 2004; Raman et al. 2003; Reid et al.
1971; Tang et al. 2000; Wang et al. 2006b, 2007), rye (Zhang and Jessop 1998), oats
(Wight et al. 2006), sorghum (Gourley et al. 1990; Magalhaes et al. 2004), and
maize (Moon et al. 1997; Rhue et al. 1978). Most of the cereals display a range of
genetic variation for Al resistance, which seems to be under control of different
alleles conditioning different levels of Al resistance (Minella and Sorrells 1992;
Raman et al. 2005). However, multigenic inheritance for Al resistance has been
observed in wheat, barley, rice, and maize (Berzonsky 1992; Echart et al. 2002;
Lima et al. 1992; Nguyen et al. 2001, 2002; Ninamango-Cardenas et al. 2003;
Raman et al. 2005). Aniol and Gustafson (1984) associated chromosome arms 6AL,
7AS, 2DL, 3DL, 4DL, 4BL, and 7D with Al resistance in the wheat landrace
cultivar “Chinese Spring.” A single gene with multiple alleles conditioning various
degrees of Al resistance appears to be common in wheat, maize, and rice (Nguyen
et al. 2001; Raman et al. 2008c; Sibov et al. 1999).
Al resistance and malate efflux has been reported to be under the control of a
single gene in wheat populations derived from ET3/ES3 (Delhaize et al. 1993a),
Diamondbird/Janz and CD87/Currawong (Raman et al. 2005), and locus XME

involved in malate efflux and Alt gene for aluminum resistance has been colocated
on the long arm of chromosome 4D (Raman et al. 2005) where Al resistance has
been mapped in other populations (Luo and Dvorak 1996; Riede and Anderson
1996). Besides one major QTL on chromosome 4DL, two additional QTLs located
on 5AS and 2DL and one region located on chromosome 7AS were identified to
contribute Al-resistance in Chinese Spring (Ma et al. 2006; Papernik et al. 2001). In
the wheat cultivar “Atlas66,” a minor QTL was located on chromosome 3BL (Cai
et al. 2008; Zhou et al. 2007).
Rye is an obligate outcrossing species in which Al resistance is conditioned by at
least four major independent and dominant loci, Alt1, Alt2, Alt3, and Alt4, located
on chromosome arms 6RS, 3R, 4RL, and 7RS, respectively (Aniol 2004; Aniol and
Gustafson 1984; Camargo et al. 2000; Collins et al. 2008; Gallego and Benito 1997;
Gallego et al. 1998a, b; Matos et al. 2005; Miftahudin et al. 2002, 2005).
Triticale, the hybrid between wheat and rye, has considerable variation for Al
resistance. Zhang et al. (1999) investigated genetic variation in root regrowth
characteristics among eight triticale genotypes using root regrowth, following Al
stress. Highly significant variation due to both general combining ability and
specific combining ability effects indicated that both additive and nonadditive
effects were important in explaining the genetic variation for Al resistance. The
high estimates of heritability and the predictability ratio for root regrowth
revealed the preponderance of additive genetic variance in the inheritance of Al
resistance.
Al resistance genes have been reported to be dominant across a range of Al
concentration in wheat (Delhaize et al. 1993b; Kerridge and Kronstad 1968).
However, incomplete transfer of genes for aluminum resistance has been reported
in wheat (Delhaize et al. 1993b). Tang et al. (2002) observed that neither wheat
cultivars “Centuary-T” nor “Chisholm-T,” which each contain an Al resistance
genes from “Atlas 66,” possessed the same level of Al resistance as “Atlas 66,” as
previously suggested (Johnson et al. 1997). Similarly, the reduction of Al resistance
genes from rye when they are present in a wheat background was observed. Aniol
and Gustafson (1984) suggested that “some genes” are acting as modifiers and thus
alter the expression of Al resistance gene. Aniol and Gustafson (1984) also reported
that the loss of number of different wheat chromosome arms reduced Al resistance.
The loss of the short arm of wheat chromosomes 5A or 7A, or the long arm of
chromosome 4D, resulted in a much lower rate of Al-induced malate release from
the root apex (Tang et al. 2002). These findings suggest that there are loci located on
wheat chromosomes 5A and 7A that have the capacity to modify the expression of
Al resistance via malate efflux. Besides one major QTL located on wheat chromo-
some arm 4DL, two additional QTLs located on wheat chromosome arms 5AS and
2DL and one region located on wheat chromosome arm 7AS were identified to also
contribute to Al resistance (Ma et al. 2006).
It has been documented that Al resistance is a dosage-dependent trait (Minella
and Sorrells 1997); therefore, there is a need to develop varieties resistant to high
concentration of Al.
11.5 Molecular Mapping of Al Resistance Loci
The identification of DNA markers diagnostic for Al resistance can accelerate the
development of acid-soil-resistant cultivars that can remain productive even under
Al stress. Molecular markers have proven to be efficient tools in identifying specific
loci controlling qualitative and quantitative traits including for Al resistance. Al
resistance loci have also been mapped using cytogenetical (Aniol and Gustafson
1984; Lagos et al. 1991; Minella and Sorrells 1997) and linkage mapping
approaches (see reviews Garvin and Carver 2003; Wang et al. 2007). Two methods
based upon bulk-segregant analysis and QTL mapping (Ma et al. 2004; Magalhaes
et al. 2004; Nguyen et al. 2001, 2002, 2003; Ninamango-Cardenas et al. 2003;
Raman et al. 2002, 2005; Tang et al. 2000; Wu et al. 2000) have been used
predominantly for locating loci associated with Al resistance in cereals (Table 11.1).
Marker-trait linkages are typically determined by linkage and QTL mapping
approaches utilizing F2, DH, or recombinant inbred line (RIL) populations devel-
oped from contrasting-phenotypic parental genotypes. Al resistance loci
(Table 11.1, Fig. 11.2) have been tagged using molecular markers based upon
randomly amplified polymorphic DNA-RAPDs (Loarce et al. 1996; Masojć et al.
2001; Philipp et al. 1994; Senft and Wricke 1996), restriction fragment length
polymorphism-RFLPs (Riede and Anderson 1996; Tang et al. 2000), simple
sequence repeat-SSR (Cai et al. 2008; Ma et al. 2005; Masojć et al. 2001; Raman
et al. 2001, 2002, 2006; Saal and Wricke 1999; Wang et al. 2007), amplified
fragment length polymorphisms-AFLP (Miftahudin et al. 2002; Raman et al.
2002; Wu et al. 2000), diversity arrays technology-DArT techniques (Wang et al.
2007; Wenzl et al. 2006), and candidate gene markers (Fontecha et al. 2007; Raman
et al. 2005, 2008c; Wang et al. 2007) in biparental populations.
Table 11.1 Wheat and Aegilops tauschii genotype by Al resistance phenotype, TaALMT1 coding
allele, and GenBank accession number (adapted from Raman et al. 2005)
Genotype Phenotype* Allele NCBI genbank accession no.
“ET8” Al-res TaALMT1-1 DQ072260
“Tasman” Al-res TaALMT1-1 DQ072270
“Diamondbird” Al-res TaALMT1-1 –
“Halberd” Al-res TaALMT1-2 DQ072265
“Chinese Spring” Al-res TaALMT1-2 DQ072262
“Maringa” Al-res TaALMT1-2 DQ072267
“Embrapa” Al-res TaALMT1-1 DQ072264
“Currawong” Al-res TaALMT1-2 –
“Cranbrook” Al-sens TaALMT1-1 DQ072263
“Spica” Al-sens TaALMT1-2 DQ072268
“Sunco” Al-sens TaALMT1-2 DQ072269
“Janz” Al-sens TaALMT1-2 DQ072266
“CD87” Al-sens TaALMT1-2 –
“ES8” Al-sens TaALMT1-2 DQ072261
Aegilops tauschii Al-sens TaALMT1-1 DQ072271
Al-res* and Al-sen* refer to Al-Resistant and Al-Sensitive, respectively
a b c d e f BMAG490
AMAL2
1.3 BMAG353
AMAL5
PAGA-MATA 1.4 HVGABP
3.4 Alt3
RZ448 RZ448 BMAC84 1.4 GWM608 0.7 BCD1230 0.1 Alp
15.2 BMAC181 4.3 CDO1395 0.4 0.1 HVMATE
2.2 5.5 GDM125 AMAL4 0.3
HVM3 1.1 AMAL1 ABG715
5.4 BMAG353 4.8 AltBH 2.9 GWM165
1.0 1.1 AMAL3
WG464 BCD1230 BMAC310
2.1 6.2 PSR914
2.1 CDO1395
3.2 Alp PSR1051
1.1 BCD1117
4.4 HVM68
BMAC310
EBMACC9
104.8
111.9
RG191
RZ745 13.1
12.7 CDO1395
RG391
0.6 CDO1395 15.7
ACC-CTG2
Fig. 11.2 Location of qualitative and quantitative loci conditioning Al tolerance in wheat, barley,
rice, and rye that were mapped on homologous group 4 chromosomes. Bold letters indicate
position of loci associated with Al tolerance. (a) IR64/O.rufipogon (Nguyen et al. 2002),
(b) IR1552/Azucena (Wu et al. 2000), (c) Dayton/Harlan Hybrid (Raman et al. 2003), (d) Milla
and Gustafson (2001), (e) Miftahudin et al. (2002)¼Alt4 on 7RS and (f) Dayton/Gairdner (Wang
et al. 2007)
Association mapping can be utilized for investigating linkage disequilibrium

close to Al resistance gene. This technique allows the utilization of germplasm and
advanced breeding lines rather than structured segregating populations, which
allows for genes associated with traits of interest to be identified by correlating
phenotype (Al resistance) with specific alleles at the linked loci. Most of the
breeding programs have phenotyping data with Al resistance of the breeding
populations/germplasm. Genotyping can be performed using whole genome scan-
ning at marker loci and then correlating with performance of plants under Al stress
(e.g., acid soils/nutrient solution).
11.5.1 Rye and Triticale
At least four independent and dominant loci associated with Al resistance, Alt1,
Alt2, Alt3, and Alt4, located on chromosome arms 6RS, 3R, 4RL, and 7RS,
respectively, have been described (Aniol 2004; Aniol and Gustafson 1984; Collins
et al. 2008; Fontecha et al. 2007; Gallego and Benito 1997; Gallego et al. 1998a; Ma
et al. 2000; Matos et al. 2005; Miftahudin et al. 2002, 2004). Previously, Alt3 was
mapped to the long arm of chromosome 4R (4RL) in the population derived from
M39A-1-6 M77A-1 (Miftahudin et al. 2002, 2004, 2005). More recently, Collins
et al. (2008) confirmed that Al resistance is controlled by an Alt4 locus that maps on
the short arm of chromosome 7R (7RS) instead of 4RL (Alt3) in the mapping
population from the M39A-1-6 M77A-1 cross. This location is consistent with a
previous report of Benito et al. (2009). Ma et al. (2000) compared 3DS.3RL
translocation line (ST22) and a nonsubstitution line (ST2) of triticale for aluminum
resistance and suggested the location of Al resistance gene on the short arm of
triticale chromosome 3R.
11.5.2 Barley
The cultivar “Dayton” is one of the most Al-resistant genotypes (Minella and
Sorrells 1992), and a single locus (Alp) on the long arm of the chromosome 4H
(4HL) conditions Al resistance in different “Dayton” derived populations (Raman
et al. 2003; Tang et al. 2000; Wang et al. 2004a). Stølen and Andersen (1978)
reported a dominant allele at Pht locus (4HL), which controls high resistance to
acidic soils. The same locus conditions Al resistance in other populations
(Table 11.1) including those generated from “Harrington”/“Brindabella” (Raman
et al. 2001), “Yambla”/“WB229,” “Mimosa”/“WB229” (Raman et al. 2002),
“Murasakimochi”/“Morex” (Ma et al. 2004), “Ohichi”/“F6ant28B48-16” (Raman
et al. 2005), and “F6ant28B48-16”/“Honen” (Wang et al. 2006b). Minor gene
effects for Al resistance in barley have also been suggested (Echart et al. 2002;
Raman et al. 2005) and require further validation.
11.5.3 Oat
Wight et al. (2006) utilized a mapping population of diploid oat A. strigosa Schreb
derived from a cross between “CIav 2921” (Al sensitive) and “CIav 9011” (Al
resistant) and identified four QTLs. The largest QTL explained 39% of the varia-
tion, was associated with the bcd1230 marker, and is possibly orthologous to the
major gene found in the Triticeae as well as Alm1 in maize and a minor gene in rice.
A second QTL may be orthologous to the Alm2 gene in maize. Two other QTLs
were associated with anonymous markers, which together accounted for 55% of the
variation.
11.5.4 Rice
QTL studies have identified 40 Al resistance loci on all 12 rice chromosomes,

although the number and locations vary depending on the cross or species used
(Ma et al. 2002; Nguyen et al. 2001, 2002, 2003; Wu et al. 2000). Epistatic effects
have also been observed (Wu et al. 2000). However, the greatest effect on Al
resistance was associated with genomic regions on chromosome 1 and 3 (Nguyen
et al. 2001, 2002, 2003; Wu et al. 2000). One of the QTLs mapped on chromosome
12 was linked with RG9 marker, which was linked with the major QTL for
phosphorus uptake efficiency in rice (Ni et al. 1988). Recently, Chuan-zao et al.
(2004) identified QTLs for relative root length on chromosomes 1, 9, and 12 and
one QTL for root length under Al stress on chromosome 1.
11.5.5 Maize and Sorghum
In maize, at least seven QTLs associated with Al resistance have been found on
chromosomes 2, 6, 8, and 10; the number and locations varied depending on the cross
(Ninamango-Cardenas et al. 2003; Sibov et al. 1999). In sorghum, a single locus,
AltSB, was found to control Al resistance in two highly Al resistant sorghum cultivars
and was mapped near the end of sorghum chromosome 3 (Magalhaes et al. 2004).
Generally, molecular markers that map within 5 centimorgan from the target
gene are recognized as “good” markers for utilization in marker-assisted selection
crop improvement programs. However, these markers are of limited value for map-
based cloning of the Al resistance gene as it requires very high-density map of the
target gene. Furthermore, most of the mapping studies in cereal utilized very small
mapping populations to locate loci associated with Al resistance, and their linkage
with marker loci may not be very accurate. High-resolution mapping can be
achieved by selecting molecular markers flanking “target” gene of interest (Al
resistance) from low resolution mapping studies and selecting recombinant plants,
which are then selfed and their F2:3 progeny are assessed for Al resistance, from a
large F2 population comprising 1,000–3,000 individuals. The main advantage of
this strategy is to select informative genotypes and to avoid extensive cost and time
required in comprehensive and accurate phenotyping. Intercross populations are
preferred over the doubled haploid populations as they are more informative due to
more accurate recombination frequencies and are easy to generate. This strategy
has been used to construct high density map of Al resistance loci in barley, rye, and
sorghum (Magalhaes et al. 2007; Wang et al. 2007) and to clone a AltSb gene for
aluminum resistance in sorghum (Magalhaes et al. 2007). Map-based cloning
approach has been used successfully to clone Al resistance genes in cereals.
Transposon-tagging strategy has not been feasible due to the lack of an active
transposon system in key cereals except in maize.
11.6 Molecular Synteny
Comparative mapping studies using molecular markers have revealed extensive

synteny or colinearity among the genomes of rice, wheat, barley, rye, oat, maize,
and sorghum (Devos and Gale 2000). A conserved genomic region on the long arm
of group 4 chromosomes: wheat 4D, barley 4H, rye 4R and short arm of 7R, and rice
3 exhibit macrosynteny (Devos and Gale 2000; Gale and Devos 1998; Miftahudin
et al. 2004; Namuth et al. 1994; Nguyen et al. 2003; Raman et al. 2002; Rognli et al.
1992; Tang et al. 2000; Van Deynze et al. 1995; Vos et al. 1995), and all harbor loci
for Al resistance (Luo and Dvorak 1996; Matos et al. 2005; Miftahudin et al. 2002,
2004; Nguyen et al. 2003; Raman et al. 2005; Riede and Anderson 1996; Tang et al.
2000; Wang et al. 2007).
Comparative mapping of the Al resistance loci in cereals can be assessed and
validated with a set of common markers linked with different Al resistance loci. For
example, Wang et al. (2007) showed that a wheat SSR marker (GWM165-4DL)
was located 0.45 cm from the Alp locus on the long arm of barley chromosome
4H and has also been located 1.5 cm apart from BCD1117 in the cMap of wheat
chromosome 4D (http://rye.pw.usda.gov). Tang et al. (2000) reported that BCD1117
and CDO1395 markers flank the Alp locus (Fig. 11.2). Marker CDO1395 that maps
on rice chromosome 3S also explained approximately 20–40% of the genetic
variation for Al resistance in the rice and wheat populations (Nguyen et al. 2003;
Riede and Anderson 1996). The marker BCD1230 exhibited tight linkage with the
Al resistance locus Alt4 in rye (Collins et al. 2008; Miftahudin et al. 2004), and
AltBH in wheat (Riede and Anderson 1996), but was mapped 33 cm away from Alp
locus in barley. This suggests that a colinearity breakage due to DNA rearrange-
ment between the chromosomes 4H of barley and 4D of wheat (Tang et al. 2000).
Milla and Gustafson (2001) reported a high degree of synteny between wheat, rye,
barley, rice, maize, and oat in the regions around the BCD1230 locus. This gene
encodes for a ribulose 5 phosphate 3 epimerase (R5P3E) gene, which is present in
one single copy in barley, rye, rice, and wheat. Interestingly, rye marker B4, which
is tightly linked with the Alt4 locus on 7RS (Collins et al. 2008; Miftahudin et al.
2004), mapped to chromosome 2H in barley instead of the expected 4H (Gruber
et al. 2006; Wang et al. 2007). Authors suggested that multiple copies of B4 may
exist in the barley genome, or there may be some conservation of genes between
chromosomes 2H and 4R. Silva-Navas et al. (2008) reported another ScAMLT2
gene in rye that showed sequence identities with barley ALMT1 homolog HvALMT1
(Delhaize et al. 2007; Gruber et al. 2006) and maps on the long arm of chromosome
2R. This suggests that there may be multiple copies of TaALMT1 at least in
genomes of barley and rye.
If genetic mapping anchors similar traits (such as Al resistance) to the collinear
chromosomal regions in different genomes, there is a good reason to suspect that these
loci are encoded by different alleles of a single gene (Bennetzen and Freeling 1997).
Al-resistance genes from wheat, barley, and sorghum (i.e., TaALMT1, HvMATE, and
ScMATE) have been recently isolated and mapped using biparental populations – see
Fig. 11.3 (Magalhaes et al. 2007; Sasaki et al. 2004; Wang et al. 2007). TaALMT1 has
shown a complete linkage with Al resistance locus on the long arm of chromosome 4D
of wheat (Raman et al. 2005; Ma et al. 2005). Fontecha et al. (2007) reported a
TaALMT1 homolog in rye, ScALMT1, and exhibited cosegregation with Alt4 locus of
rye on 7RS, which is consistent with the expected synteny relationships between the
wheat 4DL and barley 4HL. Sequence identities between TaALMT1 gene in wheat on
LPF SSR CAPS

1 2 3 4 5 6
SPF indel
Fig. 11.3 Structure of the TaALMT1gene – Adapted from Raman et al. (2005, 2006, 2008c).
White arrows represent the six exons that are interrupted by five introns (black blocks). LPF and
SPF represent long and short promoter fragments of the TaALMT1 gene (Sasaki et al. 2004, 2006).
Locations of SSR motif (intron 3) and SNP in exon 4 are indicated with down arrows
4DL and ScALMT1 gene on 7RS indicate that both genes are orthologous. However,
TaALMT1 gene does not show any sequence identities with rice chromosome 3
pseudomolecules but showed an approximately 90% sequence identities to the rice
genes located on the long arm of chromosome 4 (Delhaize et al. 2007). This suggests
breakdown of macrosynteny among members of triticeae.
In barley, Wang et al. (2007) delineated the Alp locus to a 0.2 cm region in the
high resolution mapping population by the flanking markers HvGABP and
ABG715 on the long arm of barley chromosome 4H. This region is syntenic with
120 kb sequence on chromosome 3 (Wang et al. 2007). Within this region, there
were no orthologs of wheat TaALMT1 gene. Instead, Wang et al. (2007) identified a
gene encoding a multidrug and toxic compound extrusion (MATE) within this
syntenic region, which showed cosegregation with the Alp locus for Al resistance
(Fig. 11.3). These results clearly indicate that despite a similar chromosomal
location for Al resistance loci in wheat and barley, the genes are likely to encode
different proteins and are therefore not orthologous. In rye, ortholog of HvMATE,
ScMATE was mapped within 27.5 cm from Alt4 locus on chromosome 7RS (Collins
et al. 2008). Ryan et al. (2009) also reported a correlation between expression of
TaMATE gene with citrate efflux involved in Al resistance in an F2 population
(Egret/Carazinho) of wheat on chromosome 4B. Existence of MATE and associated
Al resistance loci on chromosomes 4BL in wheat (Ryan et al. 2009), 4HL in barley
(Furukawa et al. 2007; Wang et al. 2007), 7RS in rye (Collins et al. 2008), and 3S in
rice (Nguyen et al. 2003) indicates genetic synteny for Al resistance via citrate
efflux. Members of the MATE family were also shown to facilitate citrate efflux
from Arabidopsis and sorghum (Durrett et al. 2007; Magalhaes et al. 2007). Al
resistance locus in sorghum AltSB was not also located within the syntenic region of
group 4 chromosomes. Therefore, AltSB appears to be different from the major Al
resistance loci in the Triticeae. Intertribe map comparisons suggest that a major
Al resistance rice chromosome 1 QTL is likely to be orthologous to AltSB. In maize,
Al resistance loci have been identified on chromosomes 2, 6, and 10 (Brondani and
Paiva 1996; Sibov et al. 1999). Comparative mapping analysis indicated that the
maize QTL region bin 6.05 (Ninamango-Cardenas et al. 2003) is homoeologous to
rice chromosome 5, where Nguyen et al. (2001, 2002, 2003) mapped a QTL for Al
resistance in rice. Another QTL region (bin 8.07) of Al (Ninamango-Cardenas et al.
2003) was found to be syntenous with rice chromosome 1 and sorghum linkage
group G (Magalhaes et al. 2004).
11.7 Mechanism of Aluminum Resistance
A number of physiological and biochemical mechanisms underlying aluminum

resistance have been proposed; see reviews (Kochian 1995; Larsen et al. 1998;
Moroni et al. 1991; Pellet et al. 1996). Ma et al. (2001) proposed two main
mechanisms of Al resistance (1) external resistance mechanisms, by which Al is
excluded from plant tissues, especially the symplastic portion of the root meristem;
and (2) internal resistance mechanisms, allowing plants to tolerate Al3+ in the plant
symplasm where Al that has permeated the plasmalemma is sequestered or con-
verted into an innocuous form. The details of these mechanisms are reviewed in a
separate chapter of this book (see Kochian). Among different mechanisms, Al-
activated exudation of low molecular weight organic acids (malate, citrate, and
oxalacetate) from root tips is now reasonably well-understood (Table 11.2) and has
been tested in an array of germplasm (Furukawa et al. 2007; Miyasaka et al. 1989,
1991; Raman et al. 2008c; Zhao et al. 2003). For example: Al-resistant wheat
genotypes release greater amounts of malate from their root apices as compared
to Al-sensitive wheat (Christiansen-Weniger et al. 1992; Delhaize and Ryan 1995;
Delhaize et al. 1993b; Raman et al. 2005, 2008c; Rincon and Gonzales 1992;
Snowden and Gardner 1993; Tang et al. 2002).
Al-activated efflux of organic acids is hypothesized to protect the root apices
from Al toxicity by chelating and detoxifying the harmful Al3+ cations in the
apoplasm or in the soil adjacent to the root apices, the most sensitive part of
the root system (Aniol 1996; Basu et al. 2001; Delhaize and Ryan 1995; Kinraide
et al. 2005; Miyasaka et al. 1989). This is further supported by studies showing that
Al3+ ions activate anion currents at the root apices of Al-resistant seedlings (Ryan
et al. 1997) via secretion of organic acids such as malate (Zhang et al. 2001). Al3+-
inducible resistance mechanisms in rye, and triticale, where a lag in Al-activated
Table 11.2 Examples of organic acid secreted by root apices of the key cereals
Genotype Organic acids Reference
Bread wheat Malate Ishikawa et al. (2000), Papernik et al. (2001),
Raman et al. (2005)
Citrate Ryan et al. (2009)
Barley Citrate Ma et al. (2004), Wang et al. (2007)
Rice Citrate Ma et al. (2002)
Rye Citrate and malate Li et al. (2000), (Ma et al. 2000)
Corn Citrate, malate, Pineros et al. (2002, 2005), Piñeros and
and oxalate Kochian (1999), Kidd et al. (2001),
Kollmeier et al. (2001), Mariano and
Keltjens (2003), Pellet et al. (1995), Wang
et al. (2004b)
Oat Citrate, malate Zheng et al. (1998a)
Sorghum Citrate Magalhaes et al. (2007)
Triticale Citrate and malate Stass et al. (2008), Ma et al. (2000)
Buckwheat (Fagopyrum Oxalate (Ma et al. 1997), Zheng et al. (1998b)
esculentum Moench.)
organic acid efflux and the rate of exudation increases over the first 12–24 h of Al
exposure, has been reported (Ellis et al. 2000). However, in wheat, malate exuda-
tion is rapidly activated by Al exposure and the rate of efflux does not seem to be
increase over time. This is further supported by the presence of different organic
acid transporters such as TaALMT1 (Delhaize et al. 2004), HvMATE/HvAACT1
(Furukawa et al. 2007), and SbMATE (Caniato et al. 2007) in wheat, barley, and
sorghum, respectively. In addition, other genes such as cysteine synthase have been
implicated in Al response in rice. More recently, Ryan et al. (2009) reported a
TaMATE gene associated with citrate efflux at least in two populations of wheat
derived from “Carazinho” – an Al-resistant wheat cultivar from Brazil. This gene
was located on the long arm of chromosome 4B. Above evidence suggest that Al
resistance is a multigenic trait.
11.8 Functional Genomic Approaches in Elucidating and

Validating Al Resistance Mechanisms
Whole genome sequencing approaches have allowed sequencing the genomes of

more than ten plant species including poplar and papaya. Wheat and barley
genomes are being sequenced under international consortia and will provide
insights into gene functions, evolution, and the origin of different cultivars. Further
research in genomics, sequencing, and bioinformatics platforms will enable us in
deciphering and manipulating the aluminum resistance in key cereals. Some of
these advancements on gene discovery, high-throughput gene expression using
microarray, altering gene expression by transformation technologies, and func-
tional characterization of Al resistance genes are discussed below:
11.8.1 TaALMT1 Gene Family
Various molecular and physiological studies have provided evidence that organic
acid efflux and internal detoxification are the key mechanisms in Al resistance in
cereals. During the last 5 years, significant advancements have been made in the
discovery of candidate functional genes for Al-resistance such as TaALMT1 (origi-
nally named ALMT1) in wheat (Sasaki et al. 2004, 2006; Yamaguchi et al. 2005),
HvAACT1/HvMATE in barley (Furukawa et al. 2007; Wang et al. 2007), and
MATESb in sorghum (Magalhaes et al. 2007). ALMT1 members facilitate transport
of malate in wheat and rye (Collins et al. 2008; Sasaki et al. 2004), whereas MATE
proteins transport citrate in Arabidopsis, barley, rye, and sorghum (Furukawa et al.
2007; Wang et al. 2007; Magalhaes et al. 2007; Collins et al. 2008).
TaALMT1 encodes a membrane-localized transporter (Yamaguchi et al. 2005)
that facilitates an Al-activated malate efflux. This gene has been isolated and
a b c d B1
e f g
VIP BMAG490 ISU52.2
KIP 1.3 BMAG353
WMC52 bPB-7502 CTG29
0.9 SCARQ4-283 2.0 UKO 1.4 HvGABP 0.6 AltSb OSJNBb0079B22
WMC48b bPB-8445 NIC 0.1 Alp 0.2
1.0 SbMATE
7.1 3.0 ENDO 0.1 HvMATE
Alt 1.0 DOF 0.3 ABG715
T755
TaALMT1 15.0 1.0 BCD1230 2.9 GWM165
M181
3.0 ME 2.0 Alt4 BMAC310
WMC331 4.0 B25
SCIM823-853 B5 27.9
SCIM881-1422 GPC
8.0 B4
OPO2-984 OSJNBa0014O06
STP
2.0 SCIM811-1376 OSJNBa0021B21
1.0 OPO2-633 OSJNAa0021B21
1.0 OPO7-666 0.3 OSJNBb0085F02
1.0 SCIM819-1434 27.5 0.3 OJ1193C08
57.6 1.0 OPQ4-725 0.6
0.9 OSJNBb0090D11
2.0 SCIM812-626
0.5 OsMATE
1.0 Alt4 OSJNAa0090D11
2.0 ScALMT1 OSJNBa0024O21
6.0 OPQ4-578 OJ1743A09
SCIM812-1138 GAB
6.0 ScMATE
SCIM823-2826
bPB-2219
0.5 bPB-9992
0.3 BMAC93
1.1 B4
2.5 HvALMT1
bPB-4377
Fig. 11.4 Molecular mapping of the major genes conditioning Al tolerance in wheat, barley, rye,
and Sorghum. (a) Diamondbird/Janz (Raman et al. 2005), (b) Dayton/Zhepi 2 (Wenzl et al. 2006;
Gruber et al. 2006; Wang et al. 2007), (c) Ailes/Riodeva (Fontecha et al. 2007), (d) Dayton/
Zhepi 2 (Wang et al. 2007), (e) (Magalhaes et al. 2007), and (f) Physical map of rice (http://www.
tigr.org), validated on 31st Jan 2008
characterized from different wheat genotypes (Raman et al. 2005; Sasaki et al.
2004). Molecular analysis has indicated that the TaALMT1 locus harbors two
alleles: TaALMT1-1 and TaALMT1-2 (Table 11.1). These alleles differ by six
nucleotides of which only two nucleotides encode for different amino acids in the
predicted protein (Sasaki et al. 2004).
The coding region of TaALMT1 is interrupted by five introns ranging from 0.1 to
1.8 kb (Fig. 11.4). TaALMT1 possesses at least 44 SNPs or small insertions/
deletions (InDels) (Raman et al. 2005). These polymorphisms in the introns are in
addition to six SNPs in the exons. Two of the six SNPs located in exons result in
amino acid changes in the predicted protein, and one of these, in exon 4, was used to
develop a CAPS marker to distinguish TaALMT1-1 from TaALMT1-2 (Sasaki et al.
2004). The intron 3 region is the largest and shows considerable allelic variability
(Raman et al. 2005, 2006, 2008c). These variations have been reported to be due to
simple sequence repeat motifs (SSR) with variable copy numbers and InDels.
Upstream and downstream sequence of the TaALMT1 was characterized to
identify allelic variations in 69 wheat lines (Sasaki et al. 2006). The first 1,000 bp
downstream of TaALMT1 was conserved among the lines examined apart from the
presence of a transposon-like sequence, which did not correlate with Al resistance.
However, the first 1,000 bp upstream of the TaALMT1 coding region was more
variable and six different promoter patterns could be discerned (types I–VI). Type I
had the simplest structure, while the others had blocks of sequence that were
duplicated or triplicated in different arrangements (Sasaki et al. 2006). Besides
six promoter patterns, allelic variants were also reported recently in highly diverse
germplasm comprising wheat cultivars, subspecies, and landraces of wheat (Raman
et al. 2008c).
11.8.2 Homologs and Paralogs of TaALMT1
Given that TaALMT1 encodes Al-activated malate transporter that facilitates Al-
activated malate exclusion in roots, it is quite likely that other Al-resistant plant
species that secrete organic acids from root apices may harbor “similar” gene.
Molecular analysis data has revealed that TaALMT1 homologs exist in Arabidopsis,
wheat, barley, maize rye, lupin, and Brassica.
In barley, Gruber et al. (2006) identified a TaALMT1 homolog HvALMT1.
Recently, Fontecha et al. (2007) identified a homolog to wheat TaALMT1 in rye,
ScALMT, at the Alt4 locus for Al resistance on chromosome 7RS in rye. This gene
encodes protein with 86% identity to TaALMT1. PCR primers were designed from a
TaALMT1, and this enabled to clone a paralog rye gene designated as ScALMT1.
This gene was found to cosegregate with the Alt4 located on 7RS by PCR amplifi-
cation using the wheat–rye addition lines (Fontecha et al. 2007). SNP polymorph-
isms for this gene were detected among the parents of three F2 populations that
segregate for the Alt4 locus. Aluminum induces expression of ScALMT1 particu-
larly to a higher level in root apices of Al-resistant cultivar as compared to a
sensitive cultivar.
Recently, Pineros et al. (2008) cloned ZmALMT1, a maize gene homologous to
the wheat TaALMT1 and Arabidopsis AtALMT1 genes. Transient expression of a
ZmALMT1::GFP chimera confirmed that the protein is targeted to the plant cell
plasma membrane. Gene expression data as well as biophysical transport charac-
teristics obtained from Xenopus oocytes expressing ZmALMT1 by Pineros et al.
(2008) further indicated that this transporter is implicated in the selective transport
of anions involved in mineral nutrition and ion homeostasis processes rather than
mediating a specific Al-activated citrate exudation response at the rhizosphere of
maize.
A gene from Arabidopsis (AtALMT1; At1g08430) encoding a TaALMT1-like
protein is located within an Al3+ resistance QTL located on chromosome 1
(Hoekenga et al. 2006). Aluminum not only activates AtALMT1 to trigger malate
efflux but also is required to induce its expression (Gabrielson et al. 2006;
Hoekenga et al. 2006). An Al3+-sensitive mutant of Arabidopsis Columbia ecotype
with a disrupted AtALMT1 gene is reported to lose the capacity for Al3+-activated
malate efflux (Hoekenga et al. 2006).
In rapeseed Brassica napus, two TaALMT1 paralogs BnALMT1 and BnALMT2
encoding proteins with 80% amino acid sequence identity to AtALMT1 and in
Brassica oleracea (BoALMT1) were cloned (Delhaize et al. 2007; Ligaba et al.
2006). BnALMTs conferred an Al3+-activated efflux of malate and increased Al3+
resistance in tobacco cell suspensions (Ligaba et al. 2006).
11.8.3 MATE Gene Family
The multidrug and toxic compound extrusion (MATE) family proteins are pro-
posed to transport small, organic compounds (Omote et al. 2006) and are the
members of a large and complex family of transporters. The human genome also
contains MATE1 and MATE2 genes encoding MATE transporters and is reported
to transport various organic cations including toxins (Hiasa et al. 2006; Masuda
et al. 2006; Otsuka et al. 2005). In contrast to MATE genes in the bacterial and
animal kingdom, plants contain more MATE-type transporters. For example,
there are 58 MATE orthologs in the genome of Arabidopsis thaliana (Omote
et al. 2006). However, the functions of most genes are still unknown. The first
report of a plant MATE transporters concerned AtALF5, which was identified from
a mutant-defective, aberrant lateral root formation in Arabidopsis (Diener et al.
2001). Heterologous expression of AtALF5 in yeast conferred resistance to tetra-
methylammonium, suggesting that its function involved detoxification as an
efflux transporter for xenobiotics.
Recently, several MATE transporters conferring Al resistance have been
reported. Two independent studies indicated that the HvMATE gene conditions Al
resistance in barley (Furukawa et al. 2007; Wang et al. 2007). Furukawa et al.
(2007) identified essentially the same gene (HvAACT1) responsible for the Al-
activated citrate secretion by fine mapping combined with microarray analysis,
using an Al-resistant barley cultivar, “Murasakimochi,” and an Al-sensitive culti-
var, “Morex,” and found the gene to be localized in barley root tip epidermal cells.
The study utilized an F4-derived mapping population from the “Murasakimochi”/
“Morex” population (Ma et al. 2004). The Al-resistant cultivar “Murasakimochi”
secreted a large amount of citrate from the roots in response to Al while “Morex”
did not (Ma et al. 2004). Furukawa et al. (2007) performed a microarray analysis
with Barley 1 GeneChip (Affymetrix Co.) to identify up- or downregulated tran-
scripts between “Murasakimochi” and “Morex” with and without Al treatment.
This analysis identified the transcript that encodes a member of the multidrug and
toxic compound extrusion (MATE) family (Barley1 probe name: Contig9960_at).
The homolog of this gene exists on rice chromosome 3, which corresponds to
HvAACT1 in barley. In Arabidopsis, MATE family members, FRDL showed the
highest homology to HvAACT1 with 59% identity and 86% similarity. The coding
region of HvAACT1 was 1,668 bp long, and the deduced polypeptide was 555
amino acids.
The MATE gene also conditions Al resistance in sorghum. Magalhaes et al.
(2007) performed high resolution mapping of Altsb by screening 4,170 gametes
from an F2 population derived from “SC283” (Al resistant) “BR007” (Al
sensitive) and identified a gene encoding a member of the multidrug and toxic
compound extrusion (MATE) family, an Al-activated citrate transporter, as respon-
sible for the major sorghum Al resistance gene locus. Aluminum-inducible Altsb
expression was associated with induction of aluminum resistance via enhanced root
citrate exudation (Magalhaes et al. 2007).
In white lupins (Lupinus albus L.), LaMATE is involved in citrate efflux and is
highly expressed under phosphorus deficiency (Uhde-Stone et al. 2005). Lupin
secretes citrate from the roots in response to phosphorus deficiency, suggesting
that MATE is also involved in the phosphorus deficiency-induced citrate secretion.
The FRD3 – a MATE protein member is also known to be involved in iron nutrition
and conferred enhanced Al resistance, presumably due to the increase of root citrate
release (Durrett et al. 2007). AtFRD3 was reported to be involved in the xylem
loading of citrate (Durrett et al. 2007) and was localized to the pericycle and cells
internal to the pericycle cells in the roots of Arabidopsis (Green and Rogers 2004).
Collins et al. (2008) reported on the presence of a cluster of genes homologous to
the TaALMT1, at the Alt4 Al-resistance locus of an Al-resistant rye. High-resolution
genetic mapping identified two resistant lines resulting from recombination within
the gene cluster. It appears that all genes flanking the gene cluster can be excluded
as candidates for controlling Alt4 resistance, including a homolog of the barley
HvMATE Al-resistance gene. In the recombinants, one hybrid gene containing a
chimeric open reading frame and the ScALMT1-M39.1 gene, each appeared to be
sufficient to provide full Al resistance. mRNA splice variation was observed for two
of the rye ALMT1 genes, and one gene contained a ~400 bp insertion in one of its
introns.
11.8.4 Expression Analysis of MATE and ALMT1 Homologs
Although members of the ALMT and MATE families differ from one another in
sequence and structure, they confer Al3+ resistance in a similar fashion: by facil-
itating organic anion efflux from roots. Aluminum resistance in wheat relies on the
Al-activated malate efflux from root apices, which appears to be controlled by an
Al-activated anion transporter encoded by the TaALMT1 gene on wheat chromo-
some 4DL (Sasaki et al. 2006). A strong correlation between malate efflux and Al
resistance in wheat (Sasaki et al. 2006) suggested that malate efflux is the primary
mechanism for Al resistance. It remains to be established whether (1) Al upregu-
lates malate efflux by interacting with TaALMT1 protein or via other intermediate
steps involved in malate efflux and/or (2) plays the role of a promoter in relation to
gene expression and Al resistance (Raman et al. 2008a, b, c).
In rye, the ScALMT1 gene was found to be primarily expressed in the root apex
and upregulated when Al was present in the medium. Fontecha et al. (2007)
reported fivefold differences in the expression between the Al-resistant and the
Al-nonresistant genotypes. Additionally, much higher expression was detected in
the rye genotypes than the moderately resistant “Chinese Spring” wheat. These
results suggest that the Alt4 locus encodes an Al-activated organic acid transporter
gene that could be utilized to increase Al resistance in plant species. Collins et al.
(2008) reported that Al-tolerant (M39A-1-6) and Al-intolerant (M77A-1) rye hap-
lotypes contain five and two genes, respectively, of which two (ScALMT1-M39.1
and ScALMT1-M39.2) and one (ScALMT1-M77.1) are highly expressed in the root
tip, the main site of Al-tolerance/susceptibility. All three transcripts are upregulated
by exposure to Al.
In barley, the relative expression of the HvMATE gene was 30-fold greater in
“Dayton” (Al resistant) than the Al-sensitive cv. “Gairdner” (Wang et al. 2007).
HvMATE expression was significantly correlated with Al resistance and Al-acti-
vated citrate efflux (Wang et al. 2007). When expressed in Xenopus oocytes,
HvAACT1/HvMATE protein mediated the efflux of citrate, and it did not mediate
malate secretion. HvAACT1 was presumed to be localized to the plasma membrane.
Transgenic tobacco expressing HvAACT1 showed higher citrate secretion in the
presence of Al and exhibited higher resistance to Al, but the citrate secretion was
not altered in the absence of Al despite the constitutive promoter in the heterolo-
gous host (Furukawa et al. 2007).
In Sorghum, an induction of Al resistance correlated closely with an increase in
root citrate exudation over time (over the 6 day period) in Al and the incremental
increase in SbMATE expression in response to Al (Magalhaes et al. 2007). Citrate
release mediated by the SbMATE was regulated at multiple levels not only by
changes in gene expression but also by a direct effect of Al3+ on transporter activity
and/or by Al-mediated posttranslational mediation of SbMATE (Magalhaes et al.
2007). SbMATE expression in a genetically diverse sorghum panel indicated that
the variation in Al resistance was due to an allelic series at the AltSb locus.
Differences in SbMATE expression explained over 95% of the phenotypic variation
for Al resistance in the panel, providing strong evidence that SbMATE underlies
Altsb and the differences in gene expression constituted the basis for allelic varia-
tion at this Al resistance locus. Similarly, (Magalhaes et al. 2007) found a signifi-
cant correlation between SbMATE expression and Al-activated root citrate release
and between citrate release and Al resistance, suggesting that differences in expres-
sion conditions the Al resistance phenotype primarily by modulating root citrate
exudation. Instead, the level of expression of either allele appears to be the major
determinant of Al3+ resistance in wheat (Raman et al. 2005). TaALMT1 is constitu-
tively expressed in root apices and the level of expression in different genotypes
correlates positively with Al3+ resistance (Sasaki et al. 2004).
Comparisons were made among Al3+-resistant and -sensitive genotypes of wheat
to correlate the level of TaALMT1 expression with sequences upstream and down-
stream of the TaALMT1 coding region, as well as the introns (Raman et al. 2005,
2008c; Sasaki et al. 2004, 2006). Polymorphisms in the introns and downstream
sequences did not correlate with Al3+ resistance. However, the promoter region
upstream of TaALMT1 was highly polymorphic between genotypes (Raman et al.
2008a, b, c; Sasaki et al. 2006). These studies reported up to seven promoter types
in the upstream region of the TaALMT1 gene. Promoter alleles differ from one
another in a number of arrangements of tandem repeats, which are thought to
influence the level of TaALMT1 expression and Al resistance (Raman et al.
2008a, b, c). The origin of these tandem repeats is unclear but may have originated
by inadvertent replication of genomic DNA by the “rolling circle” machinery used
by some viruses and transposons (Piffanelli et al. 2004) as suggested for the Mlo
locus in barley. Promoter that possess three tandem repeats but are otherwise
identical to those with two tandem repeats as shown in a study by Raman et al.
(2008a, b, c) could have arisen by unequal cross over events during recombination. .
MATE proteins are known to facilitate citrate efflux from Arabidopsis, barley,
and sorghum (Durrett et al. 2007; Magalhaes et al. 2007; Wang et al. 2007). Wang
et al. (2007) measured HvMATE gene expression in the root apices of “Dayton”
(Al-resistant) and “Gairdner” (Al-sensitive), using qRT-PCR, and found that the
relative expression of the HvMATE gene in “Dayton” was 30-fold higher than
“Gairdner.” Expression of the HvMATE gene was correlated with Al resistance
and Al-activated citrate efflux in an F2:3-derived population from Dayton/Gairdner
(Wang et al. 2007), and the expression of HvMATE was significantly correlated
with Al resistance and Al-activated citrate efflux. Of the F2:3 families assayed,
HvMATE expression and citrate efflux were greater in the homozygous Al-resistant
families than in homozygous Al-sensitive families, while families heterozygous for
Al resistance were generally intermediate for expression and citrate efflux.
11.9 Discovery of Candidate Genes Expressed Under Al Stress
Aluminum has to affect numerous physiological parameters in order to reach the

plasma membrane and the cytosol in less than 30 min (Lazof et al. 1994). Alumi-
num may induce several genes associated with oxidative stress (Richards et al.
1998) including those regulating the organic acid pathway featuring the citrate
synthase gene (Anoop et al. 2003; Garvin and Carver 2003.; Raman et al. 2005), or
the antioxidant pathway with genes for superoxide dismutase and glutathione
peroxidase (Milla et al. 2002; Richards et al. 1998), or the pathogen defense pathway
genes such as b-1,3-glucanase and phenylalanine ammonia-lyase (Cruz-Ortega
et al. 1997; Snowden and Gardner 1993), or signal transduction genes such as
cell wall-associated receptor kinase 1 gene (Sivaguru et al. 2003), or the general
stress-responsive pathway genes such as blue copper-binding protein gene
(Richards et al. 1998; Milla et al. 2002). However, most of these genes can also
be induced by other biotic and abiotic stresses. Furthermore, identification of these
genes was based on comparisons of gene expression levels using a single genotype
under Al-stressed vs. nonstressed conditions, or between two genotypes with diff-
erent genetic backgrounds under Al-stressed conditions. Recently, a gene encoding
a putative ABC transporter (ALS3) was found to be contributing to an Al resistance
mechanism in Arabidopsis, possibly by facilitating the redistribution of absorbed
Al away from sensitive root tissues (Larsen et al. 2005). Seven different genes
termed wali1–wali7, whose expression is induced by Al stress, were isolated from
root tips of Al-treated wheat (Richards et al. 1994; Snowden and Gardner 1993).
These gene sequences exhibited high similarities to rali2 (Gallego et al. 1998b).
With the availability of various genomic tools, it is possible to study transcript
abundance of many genes simultaneously on a genome-wide scale with respect to
their structure and function. Such studies have identified and characterized a set of
genes and families identified by traditional and genomic studies (Furukawa et al.
2007; Guo et al. 2007). To understand the mechanisms of Al resistance and to
identify genes responsible for Al resistance in wheat, Guo et al. (2007) constructed
suppression subtractive hybridization libraries from Al-stressed roots for two
wheat near-isogenic lines (NILs), “Chisholm-T” (Al-resistant) and “Chisholm-S”
(Al-sensitive). Relative gene expression levels between “Chisholm-T” and
“Chisholm-S” were compared at seven time points of Al stress: 15 min to 7 days.
Twenty-eight genes including genes for Al-activated malate transporter-1, entkaur-
enoic acid oxidase-1, b-glucosidase, lectin, histidine kinase, and phospoenolpyr-
uvate carboxylase showed more abundant transcripts in “Chisholm-T” and
therefore may facilitate Al resistance. In addition, genes related to senescence
and starvation of nitrogen, iron, and sulfur, such as copper chaperone homolog,
nitrogen regulatory gene-2, yellow stripe-1, and methyl-thioribose kinase, were
highly expressed in “Chisholm-S” under Al stress. The results suggested that Al
resistance is probably coregulated by multiple genes with diverse functions in
enhancing Al resistance and protecting root growth under Al stress. The highly
expressed genes in “Chisholm-S” under Al stress may be symptomatic of root
growth repression and restricted uptake of essential nutrient elements, leading to
root senescence.
11.10 Molecular Breeding for Al Resistance Using Genetic

Transformation
Several research studies indicate that Al resistance can be manipulated using

various candidate genes either involved in organic acid biosynthesis or stress
responsive genes. For example, aluminum resistance in canola (Brassica napus)
(Anoop et al. 2003; Basu et al. 2001), Arabidopsis thaliana (Koyama et al. 2000),
tobacco (Nicotiana tabacum) (de la Fuente et al. 1997), papaya (Carica papaya L)
(de la Fuente et al. 1997), and alfalfa (Medicago sativum L.) (Tesfaye et al. 2001)
has been reported to be enhanced by increasing organic acid biosynthesis through
overexpression of citrate synthase or malate dehydrogenase genes. TaALMT1 has
shown to increase Al resistance in root of transgenic tobacco cells and barley via
Al-activated malate efflux (Delhaize et al. 2004; Sasaki et al. 2004). SbMATE
conferred an Al-activated citrate efflux that results in Al resistance in wheat and
Arabidopsis (Magalhaes et al. 2007). Furukawa et al. (2007) reported the heterolo-
gous expression of HvAACT1 in Xenopus oocytes and showed efflux activity for
14C-labeled citrate but not for malate. Overexpression of this gene in tobacco
enhanced citrate secretion and Al resistance compared with the wild-type plants.
A good correlation was found between the expression of HvAACT1 and citrate
secretion in ten barley cultivars differing in Al resistance. Findings of Wang et al.
(2007) and of Furukawa et al. (2007) suggest that HvAACT1/HvMATE Al-activated
citrate transporter conditions Al resistance in barley.
Overexpression of genes induced with Al stress has also been reported to

enhance Al resistance in Arabidopsis, tobacco, and canola (Basu et al. 2001;
Ermolayev et al. 2003; Ezaki et al. 2000; Sivaguru et al. 2003).
In highly acidic soils, toxicity of Fe2þ and Mn2þ can occur as a result of an
excess of these elements in associations with Al toxicity. Genetic variability for
Fe2þ and Mn2þ toxicities has been reported in wheat (Camargo et al. 1989, 1992,
2000; Camargo and Ramos 1989; Moroni et al. 1991). Advanced breeding lines that
showed resistance to Al3þ, Mn2þ, and Fe2þ toxicities, under acid soil conditions,
exhibited a high grain yield as compared with the control (Camargo et al. 1989,
2000). Genomic region associated with Al resistance on chromosome 1 has also
been related to the ability of the rice root to exclude excessible Fe2þ toxicity (Wu
et al. 1998). Pyramiding QTLs associated with resistance to Fe and Mn and
Al toxicity would allow to develop cereal germplasm for resistance to acid soils.
As an experimental proof, the overexpression of AtFRD3, which enhanced exuda-
tion of citrate and malate from roots of transgenic Arabidopsis, led to the higher
resistance to aluminum (Durrett et al. 2007). MATE family members are of
particular importance as they have a wide range of transport functions including
anthocyanin uptake, iron translocation, and aluminum resistance. Furthermore,
FRD3 does not require Al3þ for activation; therefore, it can be manipulated to
improve the phosphorus efficiency as outlined by Delhaize et al. (2007).
11.11 Molecular Breeding for Al Resistance Using

Marker-Assisted Selection
While genetic transformation enables us to increase Al resistance in plant species

that are generally “Al-sensitive,” DNA markers allows to fast-track genes condi-
tioning Al resistance including in transgenics. Table 11.3 describes the linkage
between markers based upon RFLP, AFLP, SSR, DArT, and SNP and Al resistance
loci in different cereals. Some of these Al resistance-marker associations have been
validated in different genetic backgrounds (Raman et al. 2002, 2005, 2008c; Wang
et al. 2006b, 2007).
Among different marker systems, SSR and SNP markers appear to be suited to
marker-assisted selection (MAS) as they are abundant in plant genome, highly
reproducible and polymorphic, more amenable for high throughput marker screen-
ings. However, the use of markers in breeding programs depends upon the cost of
phenotyping, genotyping, number of lines to screen, and time. With the revolution
of technologies for genotyping such as capillary electrophoresis and SNP-typing,
the cost of marker screening is becoming more affordable. Molecular markers for
Al resistance have been applied in various cereal breeding programs in Australia
and elsewhere and have monitored the expression of desirable alleles in genetic
backgrounds. The Department of Agriculture and Food, Western Australia
(DAFWA), is planning to release an Al resistance barley variety developed using
Table 11.3 Linkage of aluminum resistance loci with PCR-based markers suitable for marker
assisted selection in cereal crops
Screening Population Chromosome Markers location Reference
method
Barley (Hordeum vulgare L.)
RG Yambla/WB229 4HL Bmag353, Bmac310 Raman et al. (2002)
WB229/Mimosa HVM68
Harrington/ 4HL Bmag353, Bmac310 Raman et al. (2001)
Brindabella
Ohichi/ 4HL Bmag353, Bmac310 Raman et al. (2005)
F6ant28B48-
16
Dayton/Zhepi2 4HL Bmag353, HvMATE, Wang et al. (2007)
HVM68, HvMATE,
GBM1071
Dayton/ 4HL Bmag353, Bmac310, Raman et al. (2003)
F6ant28B48- HVM68
16
RRG Dayton/Zhepi2 4HL HvGABP, Bmag353, Wang et al. (2007)
HVM68, HvMATE,
GBM1071
Hematoxylin Dayton/Harlan 4HL Bmag353, Bmac310, Raman et al. (2003)
staining Hybrid HVM68
Eriochrome Dayton/Zhepi2 4HL HvGABP, Bmag353, Wang et al. (2007)
cyanine HVM68, HvMATE,
GBM1071
Dayton/Gairdner 4HL HvGABP, ABG715, Wang et al. (2007)
GWM165, Bmag353
F6ant28B48-16/ 4HL Bmag353, HVM68 Wang et al. (2006b)
Honen
Root/shoot Murasakimochi/ 4HL Bmag353 Ma et al. (2004)
fresh wt Morex
ratio
Wheat (Triticum aestivum L.)
Hematoxylin Diamondbird/ 4DL TaALMT1, WMC331 Raman et al. (2003,
Janz 2008c, 2005)
Currawong/CD87 4DL TaALMT1, WMC331 Raman et al. (2008c,
2005)
Spica/Maringa 4DL TaALMT1, GWM165 Raman et al. (2008c,
2005)
Atlas66/Century 4DL WMC125, GDM125, Ma et al. (2005)
TaALMT1
Root growth BH1146/ 4DL BCD1230, GDM125, Riede and Anderson
Anahuac TaALMT1 (1996), Milla and
Gustafson
(2001), Raman
et al. (2008c)
RRG Diamondbird/ 4DL TaALMT1, WMC331 Raman et al. (2005)
Janz
Cranbrook/ TaALMT1 Raman et al. (2005,
Halberd 2008)
Sunco/Tasman TaALMT1
(continued)

Screening Population Chromosome Markers location Reference
method
Raman et al. (2005,
2008)
Atlas66/Century WMC125, GDM125, Ma et al. (2005)
TaALMT1
Atlas66/ 4DL TaALMT1, WMC331, Zhou et al. (2007)
Chisholm GDM125
3BL BARC164 Zhou et al. (2007)
FSW/ND35 4DL TaALMT1 Cai et al. (2008)
3B BARC164, BARC344 Cai et al. (2008)
2A GWM515, GWM296 Cai et al. (2008)
Carazhino/EGA- 4BL GWM495, GWM513 Ryan et al. (2009)
Burke
Rice (Oryza sativa L.)
RG, RRG IR64/O rufipogon QTLs RFLP/SSR markers (Nguyen et al. 2003)
CT9933/IR62266 QTLs RFLP/SSR markers (Nguyen et al. 2002)
Rye (Secale cereale L.)
RG,RRG Ailes Riodeva 6RS ScR01600, ScB15790 Gallego and Benito
(Alt1) (1997)
(AR6-17, AR1- SCR01600 Gallego et al.
13) (1998a, b)
Ailes Riodeva 4RL ScOPS17705 Benito et al. (2009)
(Alt3)
M39A-1- 7RS B1, B4, B11, B25, B26, Collins et al. (2008),
6 M77A-1 B27, BCD1230 Miftahudin et al.
(Alt4) (2002, 2004,
2005)
Ailes x Riodeva 7RS B1, B4, B26, Benito et al. (2009),
(Alt4) ScALMT1, Fontecha et al.
(2007)
Oats (Avena sativa L.)
CIav2921/ – SCA08 and calretB1_3 Wight et al. (2006
CIav9011 #646)
Maize (Zea mays L.)
L53/L1327 QTLs SSR Ninamango-
Cardenas et al.
(2003)
RRG/RRE Relative root growth, RG/RE Root growth, RFLP Restriction fragment length
polymorphism
MAS (Reg Lance, per communication). So far, no epistatic or pleiotropic effects of

Al resistance loci are known except in rice. Major genes/QTL effects for Al
resistance can be tested via the association mapping approach. It is expected that
advanced breeding lines and cultivars will have high linkage disequilibrium as
compared to wide diverse germplasm, and that flanking markers will be better
suitable for MAS as compared to single markers associated with the trait of interest.
Potential MAS schemes include selection of the parental genotypes for making
crosses, allele enrichment among F1 individuals, selection of marker alleles during
recurrent backcrossing and intercross populations, and genome-wide selection for

restored background genotype. The usefulness of markers in the breeding programs
will depend upon polymorphic content (PIC) value of the markers. The higher the
PIC, the more useful will be linked markers, which makes it important to test the
polymorphism on a suite of markers linked with the locus of interest. In order to
increase selection efficiency of MAS for Al resistance, diagnostic markers based on
functionally associated variation in the candidate genes for Al resistance, such as
TaALMT1, HvMATE, and SbMATE, have been developed, but unfortunately, some
of these markers were not found to be “diagnostic” (Raman et al. 2005). Nevertheless,
these candidate gene-based functional markers are preferred for association-
mapping studies and MAS as compared with whole genome marker scans. Associ-
ation mapping approach circumvents the need for construction of linkage maps and
linkage analysis in the biparental populations.
11.12 Allele Mining
The gene pool of cereals present in nature has tremendous allelic variation for
different traits of agronomic importance. Considerable genetic variation for Al
resistance also exists in all key cereals including wheat, rice, and barley (Aniol
1996; Bona et al. 1993; Camargo et al. 1991; Caniato et al. 2007; Ceretta 1988;
Mazzocato et al. 2002; Minella and Sorrells 1992; Mugwira et al. 1976; Raman
et al. 2008c; Reid et al. 1969; Stodart et al. 2007; Wu et al. 2000; Xu et al. 2004). An
array of molecular techniques is available to detect and understand the overall
diversity for Al resistance genes within species including RFLP, RAPDS, SSRs,
DArT, AFLP, and SNP. Furthermore, genomic approaches also provide the mean to
access genes directly via gene discovery programs. Functional gene-specific mar-
kers are more suited for allele mining, as they are functionally relevant directly to
the trait of interest.
Among various cereals, at least three candidate genes, TaALMT1, HvMATE, and
SbMATE, have been correlated with Al resistance in wheat, barley, and sorghum,
respectively, and could be utilized to determine allelic diversity within the genes
conditioning Al-resistance. Raman et al. (2008a, b, c) characterized more than 300
genotypes of wheat for aluminum resistance using a two-step approach that involves
(1) screening of germplasm using hematoxylin staining method, and (2) reevalua-
tion of Al-resistant germplasm using the TaALMT1 gene-specific markers for exon 4
(Sasaki et al. 2004), intron 3 (Raman et al. 2006), and long/short promoter sequence.
Analysis of TaALMT1 exon sequences has identified two alleles neither of which is
diagnostic of Al resistance (Sasaki et al. 2004; Raman et al. 2005). By contrast,
intronic regions display significant polymorphisms (Raman et al. 2005). Among the
different introns, three regions show considerable allelic variability (Raman et al.
2006). These variations are due to SSR motifs with variable copy numbers and
Indels (Raman et al. 2005; 2006). These markers identify at least eight alleles
(Raman et al. 2006, 2008a, b, c). Analysis of upstream region of TaALMT1 (Sasaki
et al. 2006) revealed at least six types of promoter region (Sasaki et al. 2006).
Raman et al. (2008c) utilized TaALMT1 gene-specific markers to characterize

over 400 cultivars, landraces, and subspecies of bread wheat and found that at least
23 haplotypes. Among different haplotypes, promoter V was present in most of the
Al-resistant germplasm. Correlation of gene haplotype structure and phenotypic
variation provided the basis for a new paradigm in wheat marker-assisted breeding
based on direct selection of superior alleles. Magalhaes et al. (2007) also found that
the large differences in aluminum resistance in sorghum are largely due to an allelic
series at the AltSb. Molecular markers have proved to be useful in understanding the
origin and distribution of Al resistance. Raman et al. (2008c) demonstrated that
markers based on TaALMT1 intron, exon, and promoter regions can trace the
inheritance of the Al resistance locus within wheat pedigrees and track Al resistance
in breeding programs. Molecular and pedigree analysis suggested that Al resistance
in modern wheat germplasm has been derived from several independent sources
and that most of the promoter alleles associated with Al resistance preexisted in
Europe, the Middle East, and Asia prior to the dispersal of domesticated germplasm
around the world.
Genetically diverse sorghum accessions indicated that the Al resistance related
mutations are located in regulatory regions of AltSb, and this may be due to
regulatory MITE sequences (Magalhaes et al. 2007). Further analysis of sorghum
near isogenic lines indicated that significant allelic variation occurs at the AltSb loci
for the lines in the 1.9 kb MITE insertion class, and they have been shown to possess
alleles that encode significant different Al resistance levels (Caniato et al. 2007).
For plant species that do not display significant variability for aluminum resis-
tance such as barley, rice, and durum wheat, there is an urgent need to broaden the
gene pool for enhancing Al resistance. It is well known that wild species and
landraces have unique alleles that are not found in the cultivated gene pool and
can be especially potent sources of abiotic stress resistance traits (Ellis et al. 2000).
Discovery of such alleles in landraces and wild progenitors such as Aegilops
uniaristata, and A tauschii, conditioning Al resistance, would provide new means
to develop varieties suitable for cultivation on acidic soils. Novel alleles from
resistant landraces/wild species can be introgressed/backcrossed into adapted
high-yielding genetic backgrounds to ensure “optimum” yield required for local
adaptation.
11.13 Conclusions
Significant achievements have been made in the identification and utilization of

genetic variability for Al resistance in cereals. Thousand years of untargeted
selection by early farmers and targeted selection by the modern breeding programs
have narrowed down genetic variation in cereal germplasm. Genepools including
landraces and wild relatives need to be exploited by screening, intercrossing, and
subsequently introgressing desirable gene complexes, minus any associated linkage
drag, into the target species. Further efforts need to be focused upon screening plant
germplasm for better sources for Al resistance, identifying new sources of Al

resistance, understanding origin and transmission of superior alleles from cultivated
and wild relatives, and understanding other mechanisms involved with Al resis-
tance (besides organic acid efflux) and regulatory networks associated with Al
resistance, in conjunction with value-added trait genes involved in yield and other
abiotic stresses functioning under acid soil conditions. New data on regulatory
pathways involved in Al stress response generated using functional genomic
approaches is becoming available and may be useful to develop and enhance
level of Al resistance in crop species. There is no doubt that genomics-assisted
breeding will accelerate the development of stable Al-resistant crop varieties. A
higher degree of Al resistance might also be achieved by pyramiding multiple
copies of gene complexes conditioning Al resistance (such as 4DL, 4BL, 3BL,
and 2AL in wheat) into selected germplasm and/or by genetic manipulating expres-
sion of endogenous genes or by expressing foreign genes in desired germplasm. In
order to make MAS and GMO approach more effective, careful establishment of
breeding strategy is required.
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Chapter 12
Molecular Breeding of Rice for Problem Soils
Abdelbagi M. Ismail and Michael J. Thomson
Contents
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
12.2 Abiotic Stresses Affecting Root Growth in Problem Soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
12.2.1 Salt Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
12.2.2 Mineral Deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
12.2.3 Mineral Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
12.3 Current and Future Prospects of Marker Assisted Backcrossing for Breeding
Varieties Adapted to Problem Soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
12.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
12.1 Introduction
Problem soils are globally widespread and seriously constrain agriculture produc-
tion. These soils generally contain toxic amounts of minerals or are deficient in
some essential plant nutrients. They are generally of limited agricultural use
because of variable factors, including toxic levels of salts or elements such as
iron, aluminum, and heavy metals, as well as deficiency in other essential nutrients,
such as phosphorus, iron, and zinc. Both acid and alkaline soils have low produc-
tivity. Globally, acid soils constitute about 2,500 Mha, of which over 1,700 Mha are
in the tropics. These soils provide great potential for agriculture expansion if
effectively utilized. Soil acidification problems are also likely to increase with
rising CO2 levels in the atmosphere, continuous use of ammonium-based nitroge-
nous fertilizers, removal of nutrients in farm products without replenishment, and
nitrate leaching. The highly weathered acid soils of the tropics are inherently low in
productivity with high Al and Fe and low in phosphorus.
A.M. Ismail (*) and M.J. Thomson

International Rice Research Institute (IRRI), DAPO 7777, Metro Manila, Philippines
e-mail: abdelbagi.ismail@cgiar.org

290 A.M. Ismail and M.J. Thomson
Problem soils constitute a considerable proportion of rice production areas,

which are mostly inhabited by poor communities because of limited opportunities.
Vast areas of lands suitable for rice production in South and Southeast Asia, South
America, and Africa are currently underused or entirely unused because of these
soil problems; however, these soils remain potential targets for extra food produc-
tion and can significantly contribute to the elimination of global hunger and poverty
if sufficiently exploited. For example, in rice-producing areas, high-yielding vari-
eties with sufficient tolerance of predominant soil problems are expected to provide
a yield advantage of over 2 tons per hectare on these problem soils (Ponnamperuma
1994).
Problem soils bring about their primary effects on plant growth and productivity
essentially via plant roots. These effects either directly or indirectly suppress root
growth, with considerable negative impacts on water and nutrient uptake, as well as
on plant growth and productivity. Mineral toxicities (excess salts, soil acidity, Fe
and Al toxicity, and numerous heavy metals) as well as deficiencies (Zn, P, and Fe)
can have direct effects on root growth. These factors can also lead to indirect
responses in growth and yield exerted through roots in problem soils, such as
excessive uptake of cations and anions when these elements are in abundance,
causing toxic effects in relatively more sensitive plant tissues as young leaves,
growing tissues, and reproductive organs. Moreover, even mild soil problems can
result in chemical and/or hydraulic signals, which induce responses in shoots that
can negatively impact growth and productivity. Multiple abiotic stresses are com-
monly experienced in these soils, such as P- and Zn-deficiencies and Fe and
Al-toxicities in acid and alkaline soils. Tolerance of these stresses involves a
plethora of complex traits and mechanisms, and this complexity has slowed previ-
ous breeding efforts to develop high-yielding varieties with sufficient adaptation to
such conditions (Ismail et al. 2007). These challenges forced breeders to search for
innovative strategies to make further progress on the seemingly intractable pro-
blems that have continued to hamper conventional breeding efforts. New
approaches are necessary to genetically dissect and incorporate these complex
adaptive traits into high-yielding rice varieties while simultaneously retaining
their good agronomic and quality attributes. This could be achieved if the genes
responsible for tolerance of these stresses were identified and exploited for crop
improvement using modern breeding and biotechnological approaches.
Recent developments in genomics and molecular biology and the advances in
molecular marker techniques have made it possible to unravel the genetic determi-
nants of complex traits underlying stress tolerance in crop plants. Genetic linkage
mapping of polygenic traits has led to the identification of quantitative trait loci
(QTLs) that control complex traits in plants. Furthermore, by using natural genetic
variation to investigate the intricate systems plants have developed to deal with
the multitude of abiotic stresses in natural ecosystems, geneticists can now identify
superior tolerance alleles and transfer them into high-yielding varieties that are
intolerant of a particular stress. These efforts led to the development of marker-
assisted breeding systems for cultivar improvement through the transfer of
major QTLs into popular varieties and advanced breeding lines. While transgenic
12 Molecular Breeding of Rice for Problem Soils 291
approaches will ultimately play an important role in developing abiotic stress-tolerant

plants, a marker-assisted approach provides a useful alternative when the required
traits are available within the species gene pool and particularly in instances where
genetically modified food crops such as rice are still far from being widely accepted.
In this chapter, we will present cases where molecular markers are being used as
tools for dissecting complex traits associated with tolerance of abiotic stresses
encountered in problem soils. We will then provide several cases where these tech-
niques are currently being used in rice to identify and transfer major loci associated
with tolerance, particularly those that are directly or indirectly mediated through plant
roots. We also present some insights into how future advances in marker development
and high throughput genotyping could impact progress in breeding to more efficiently
develop high-yielding, stress-tolerant varieties to enhance and stabilize productivity
in problem soils as well as in other areas facing similar abiotic stresses.
12.2 Abiotic Stresses Affecting Root Growth in Problem Soils
Adaptation to unfavorable soil conditions generally involves several complex and

interrelated physiological and morphological tolerance mechanisms, most of them
expressed at the root level. Understanding the mechanisms that are involved in
these processes and how they are integrated and regulated will ultimately speed the
efforts to improve the performance of crop plants for problem soils. Traits that
allow traditional cultivars to survive and produce well under such extreme condi-
tions need to be incorporated into popular varieties and elite breeding lines, without
substantial changes in their adaptive and quality traits. This will require a system-
atic analysis of the genetics and physiology of such characters, together with a
thorough evaluation of the target environments to select for relevant traits. Mechan-
isms associated with tolerance of various abiotic stresses encountered in problem
soils can now be dissected into component elements that can then be targeted for
molecular breeding through the use of molecular markers that are linked to genes
controlling each specific trait component. Research over the past few decades
uncovered many agronomically useful characters within and between cultivated
and wild rice germplasm, making genetic improvement more feasible. We will
highlight the progress made in several abiotic stresses common to rice problem
soils, particularly those affecting root growth and function.
12.2.1 Salt Stress
Excessive salt stress is a major constraint for crop production in vast areas of the
world, affecting over 12 million ha of rice land in Asia. Salinity in coastal areas
fluctuates within the year, being high during the dry season because of tidal inundation
and intrusion from saline shallow water tables but decreasing with the freshwater flush
during the rainy season. Secondary salinization can also occur as a result of misuse
of irrigation water with poor drainage, and recently this has become an alarming
problem in inland areas worldwide, steadily leading to soil deterioration and eventual
abandonment by poor farmers. About 10 million ha of agricultural lands in the world
are believed to be lost annually to salinization (Pessarakli and Szabolcs 1999).
12.2.1.1 Salt Stress Tolerance in Rice
Rice is moderately sensitive to salt stress, yet it is still preferred as an initial crop
during soil reclamation because of its unique ability to thrive in standing water.
Sensitivity also varies with the climate and the stage of development, with poor
association between tolerance at the two most sensitive stages, early seedling, and
reproduction (Moradi et al. 2003; Peng and Ismail 2004). Considerable genetic
variation in salinity tolerance was reported in rice (Flowers and Yeo 1981), and
progress has been made in developing elite breeding lines with a reasonable level of
tolerance, some of which were released as commercial varieties (Gregorio et al.
2002; Senadhira et al. 2002; Salam et al. 2007). Salinity tolerance in rice is complex
and involves several physiological and adaptive mechanisms (Yeo and Flowers
1986; Peng and Ismail 2004; Ismail et al. 2007). The physiological bases of salt
tolerance during early seedling stage are fairly well understood, involving key traits
such as high seedling vigor to dilute salt concentration in plant tissues, selective ion
uptake by roots, compartmentation of harmful ions in structural and older tissues
(particularly older leaves, stems, leaf sheath and roots), responsive stomata that
regulate water and salt uptake in response to increasing salt stress in the rhizosphere,
high tissue tolerance through sequestering salts in the apoplast, and recirculation of
sodium back to roots to avoid accumulation of toxic concentrations in the cytoplasm.
The latter is probably achieved through a set of active processes involving a gene
family of ion transporters such as Na+/H+ antiporters that sequester salt in vacuoles
(Blumwald et al. 2000) or move it out of the cell cytoplasm and recirculate it back to
the roots (Berthomieu et al. 2003). Responsive stomata that quickly close upon
initial exposure to salt stress, probably in response to signals from roots, but partially
reopen after a period of acclimation could also contribute to tolerance by minimizing
salt uptake. Antioxidant scavenging systems also seem to play an important role
through neutralizing toxic radicals generated during stress (Moradi and Ismail
2007). Overexpression of superoxide dismutase, a key enzyme in the ascorbate–
glutathione pathway, conferred tolerance of salinity in Arabidopsis (Gao et al. 2003).
During reproductive development, tolerant genotypes also tend to exclude salt from
flag leaves and developing panicles (Yeo and Flowers 1986; Khatun et al. 1995).
12.2.1.2 Germplasm Improvement for Salt Stress Tolerance
Despite the fact that traits associated with salinity tolerance in rice are seemingly
independent, all known salt-tolerant landraces are superior in only one or a few of
them, and significant genetic variation exists for each particular trait. This suggests
the possibility of identifying better donors that can provide superior combinations
of alleles at useful genes. Combining the traits that are effective at seedling and
reproductive stages will then ensure the development of rice cultivars with higher
levels of salt tolerance. Selection could essentially be made in parallel for individ-
ual traits, which can then be combined through multiple crosses. Moreover, identi-
fying and fine-mapping major QTLs and cloning of genes underlying these traits
will particularly help speed the breeding process by precise targeting of useful
alleles using marker-assisted backcrossing (MABC). By reducing linkage drag,
MABC has allowed the precise introgression of agronomically useful traits into
popular varieties without changing their adaptive or quality traits. A good example
is the transfer of the SUB1A locus into numerous rice varieties, making them
extremely tolerant of submergence (Neeraja et al. 2007; Septiningsih et al. 2009;
Singh et al. 2009).
Considerable progress was recently made in deciphering genes associated with
salt stress tolerance in plants. For example, numerous cases demonstrated the role
of sodium transporters in maintaining ion homeostasis in plants under salt stress
through mechanisms that remove sodium from the cytoplasm by either compart-
menting it into vacuoles or extruding it out of the cell (Horie and Schroeder 2004).
The salt overly sensitive (SOS) signaling pathway was characterized in Arabidopsis
as being involved in signal perception and ion homeostasis (Zhu 2003), and the role
of this system in controlling salt stress tolerance in rice was established recently
(Martinez-Atienza et al. 2007). The HKT family of transporters was also shown to
have significant roles in sodium and potassium uptake and homeostasis in a number
of plant species including rice (Horie et al. 2001; Golldack et al. 2002), and the
cloning of the rice QTL SKC1, originally detected by its effect on K+ concentration,
identified the causal gene as the sodium transporter OsHKT8 (Ren et al. 2005).
Discovery of the genes underlying tolerance of salt stress will help in designing
functional markers for more accurate and efficient use in MABC.
Several studies have identified QTLs associated with salinity tolerance in rice
(Table 12.1). A major QTL for salt tolerance was tagged with an RFLP marker on
chromosome 7 using an F2 population derived from salt-tolerant japonica rice
mutant M-20 and the sensitive original variety 77-170 (Zhang et al. 1995). Using
a cross of an indica variety of moderate tolerance (IR64) with a sensitive japonica
variety (Azucena), seven QTLs for seedling traits associated with salt stress toler-
ance were mapped, though all explained less than 20% of the variation (Prasad et al.
2000). Using a cross between two moderately tolerant elite indica breeding lines,
one of which had Pokkali in its pedigree, several QTLs were identified, of which the
QTL with the largest effect was for K+ uptake on chromosome 9, explaining 19.6%
of the variation (Koyama et al. 2001). A study employing the highly tolerant indica
variety Nona Bokra with the susceptible japonica Koshihikari identified several
QTLs of much larger effect, including the SKC1 QTL for shoot K+ concentration on
chromosome 1 and a QTL for shoot Na+ concentration on chromosome 7 (Lin et al.
2004). Furthermore, using a population of 80 recombinant inbred lines (RILs)
generated from a cross between sensitive variety IR29 and a tolerant landrace,
Pokkali, QTLs were identified on chromosomes 1, 3, 4, 10, and 12 for salinity
294
Table 12.1 Comparison of the number of QTLs and size of the largest QTL detected across different mapping studies for tolerance to various problem soils
Trait Total QTLs Largest QTL Max. R2 Tolerant donors References
Salinity tolerance >20 Chr. 1 (Gregorio 1997) 64%a (Na/K ratio) Pokkali, IR64, Nona Bokra Zhang et al. (1995), Gregorio
(1997), Prasad et al. (2000),
Koyama et al. (2001),
Bonilla et al. (2002), Lin
et al. (2004)
P-deficiency tolerance 10 Chr. 12 (Ni et al. 1998) 61% (Shoot dry Kasalath, IR20 Wissuwa et al. (1998), Ni et al.
weight) (1998), Shimizu et al. (2004)
Zn-deficiency tolerance 6 Chr. 12 (Wissuwa et al. 24% (Mortality) Jalmagna Wissuwa et al. (2006)
2006)
Fe-toxicity tolerance 9 Chr. 3 (Wan et al. 2003) 48% (Stem dry Azucena, Kasalath Wu et al. (1997, 1998), Wan
weight) et al. (2003)
Al-toxicity tolerance >30 Chr. 8 (Nguyen et al. 2002) 28% (Ratio of root Azucena, Chiembau, CT9993, Wu et al. (2000), Nguyen et al.
length of stress O. rufipoogon, (2001, 2002, 2003), Ma et al.
vs. control) Koshihikari, Asominori (2002), Xue et al. (2006,
2007)
a
Note: This R2 value was obtained using phenotypic extremes for the QTL analysis
A.M. Ismail and M.J. Thomson
tolerance during seedling stage, including a major QTL designated Saltol mapped
on chromosome 1 explaining 64% of the variation for seedling shoot Naþ/Kþ
ratio using phenotypic extremes and 43% of the variation in a subsequent study
(Gregorio 1997; Bonilla et al. 2002). Current efforts at IRRI include fine mapping
of the Saltol QTL, using MABC to incorporate this QTL into popular varieties
sensitive to salt stress, and targeting additional QTLs for salinity tolerance at
different growth stages for combining multiple QTLs to increase the level of
tolerance in salt-stressed environments. As with the SUB1 QTL, Saltol is being
transferred into popular rice varieties using MABC to precisely incorporate the
Pokkali introgression conferring tolerance while reducing any unwanted DNA
segments that may contain negative characters.
12.2.2 Mineral Deficiency
Nutrient deficiency induced by problem soils is wide spread in rice production

areas, particularly in soils with high fixing capacity as in acid and calcareous/sodic
soils. This induced deficiency is causing considerable reductions in grain yield, and
is further being worsened by the high demand for these nutrients under the newly
evolving intensive farming systems using modern varieties. The rate of plant
nutrient removal from the soil by modern high-yielding rice varieties is about
three times that of traditional varieties (De Datta et al. 1990), which further
aggravates the problem. Phosphorus and zinc are the most widely encountered
deficiencies in rice soils caused by their immobilization in forms that are not readily
available for plant roots. Varieties with greater ability to mobilize and use these
nutrients will be more efficient in these soils, especially where farmers are resource-
poor and adding sufficient nutrients to overcome these deficiencies is out of their
reach.
12.2.2.1 Phosphorus Deficiency
Phosphorus is the most important inorganic plant nutrient after nitrogen but the
least available in soils because of its limited mobility and the tendency of most soils
to fix it into forms that are hardly available for plant roots, as in most alkaline and
acid soils. This tight binding of P in the soil rather than a low total P content is often
the primary cause of its deficiency. As a result, phosphorus deficiency is widespread
in both upland and lowland rice-growing areas. In most of these areas, phosphorus
fertilizers are not always available or affordable for resource-poor farmers and the
tendency of soils to rapidly fix it reduces fertilizer use efficiency.
Breeding cultivars capable of efficient mining of the large pool of P already
existing in most rice soils will help increase and sustain yields in low-input
agricultural systems. Large variability among lowland and upland rice cultivars in
their ability to utilize soil P was observed (Wissuwa and Ae 2001a); however, no
formal breeding program has yet been in place to develop P-efficient varieties. The
concentration of available P in soils is usually very low and coupled with its
extremely slow mobility, particularly in highly weathered soils, suggesting that
its acquisition must occur against a steep concentration gradient involving active
uptake. So far, two main types of phosphate transport systems were identified in
rice, high-affinity and low-affinity transport systems. The low-affinity transport
system appears to be expressed constitutively, whereas the high-affinity uptake
system is strongly enhanced when phosphorus is limiting (Vance et al. 2003).
In plants, two types of mechanisms are involved in P deficiency tolerance,
internal mechanisms associated with the efficient use of P by plant tissue and the
external mechanisms that allow greater P uptake by plant roots. Genetic variation in
internal P efficiency was observed in rice but is mostly associated with low P
uptake. External efficiency is probably the most important mechanism underlying
tolerance to P deficiency in rice. However, mechanisms responsible for this effi-
ciency still await further studies. The main external mechanisms observed in plants
involve (a) the ability to develop long, fine hairy roots to maximize exposure to the
rhizosphere, (b) the ability to mobilize P through pH changes or the release of
ligands or chelating agents such as organic acids, (c) the ability to utilize soil
organic P through release of phosphate enzymes, and (d) the ability to associate
with mycorrhizal fungi (Kirk et al. 1993; Hedley et al. 1994). Mycorrhizae are
expected to be less effective in fine-rooted crops such as cereals, especially in
anaerobic flooded soils.
Root Characteristics Associated with High P-Uptake
Because of its slow mobility in the soil, root morphological characteristics such as
length, surface area, fitness, and intensity of root hairs are found to be important for
P uptake in numerous crops (Otani and Ae 1996; Kirk and Du 1997). Using rice
cultivars of different origins, Wissuwa and Ae (2001a) observed a strong relation
between tolerance to P deficiency with both root size and root uptake efficiency but
with stronger association with the root size. A large root system may therefore be
adaptive and may provide a more reliable criterion to identify genotypes with
tolerance of P deficiency. The ability of rice cultivars to solubilize P fixed in the
soil has been suggested (Hedley et al. 1994; Saleque and Kirk 1995; Kirk et al.
1999). This could involve acidification of soils by roots, and changes of over two
pH units had been reported in the immediate vicinity of roots in flooded soils
(Saleque and Kirk 1995). Under aerobic soil conditions, mechanisms involved in
remobilization of P are expected to be different and could involve the secretion of
low molecular weight organic acids such as citrate (Kirk et al. 1999). Organic acids
may act as chelating agents for aluminum and iron to free P in soil solution, and
high rates of excretion of P-solubilizing organic acid anions from roots was
reported in rice in response to P-deficiency (Kirk et al. 1993).
Germplasm Improvement for Higher P-Uptake Efficiency
Genotypic differences in P deficiency tolerance in rice were reported long ago;

however, breeding efforts were limited to screening available cultivars and
advanced breeding lines for superior performance in P deficient soils rather than
developing genotypes with higher efficiency of P uptake (Fageria et al. 1988;
Hedley et al. 1994; Ismail et al. 2007). Traditional landraces are more efficient in
P uptake than modern high-yielding varieties (Wissuwa and Ae 2001a). These
landraces will therefore provide potential donors of P-deficiency tolerance for
cultivar improvement using conventional approaches and also could serve as
sources of agronomically important QTLs and genes identified through mapping
and subsequent cloning.
Tolerance of P deficiency is quantitatively inherited in rice, with both additive
and dominant genetic effects. QTLs associated with P-deficiency tolerance were
identified in two mapping studies (Wissuwa et al. 1998; Ni et al. 1998). Wissuwa
et al. (1998) used a backcross inbred population with the recurrent parent Nippon-
bare (japonica, sensitive) and the landrace Kasalath (aus, tolerant) and identified
four QTLs for P uptake on chromosomes 2, 6, 10, and 12, including a major QTL on
chromosome 12 that controls most of the variation in P-deficiency tolerance. For P
uptake, this QTL had an LOD score of 10.7 and explained about 28% of the
phenotypic variation. Ni et al. (1998), using RILs from the cross of IR20 (tolerant)
with IR55178-3B-9-3 (sensitive), found a similarly strong QTL in the same loca-
tion. They measured P uptake efficiency as relative tillering ability, relative shoot
dry weight, and relative root dry weight. Moreover, an intermediate QTL on
chromosome 6 and several other minor QTLs were mapped to several chromo-
somes. The QTL on chromosome 6 explained 25–34% of the variance for the above
traits in the Ni et al. (1998) study but had a much lower effect (R2 ¼ 9.8%) in the
field study of Wissuwa et al. (1998). The QTL on the long arm of chromosome 6
was also identified in another independent mapping study using a population
developed from a cross of the tolerant Kasalath and the intolerant Gimbozu and
was found to be associated with phosphorus deficiency-induced root elongation
(Shimizu et al. 2004). Recently, the position of this QTL, named qREP-6 for root
elongation under phosphorus deficiency, as well as its role were confirmed using
chromosome segment substitution lines developed in the background of Nippon-
bare (Shimizu et al. 2008). The substitution line carrying qREP-6 had higher
tillering ability on P-deficient soils and also higher phosphorus concentration in
the shoot, suggesting that this QTL will potentially be important in breeding
cultivars with better root traits for P-deficient soils. The qREP-6 was fine-mapped
in an F2 population and a total of 37 genes were annotated in the region (Shimizu
et al. 2008), paving the way for its subsequent positional cloning. This will further
enhance our understanding of its mechanistic role and quantify its effects in
improving adaptation to phosphorus deficiency stress.
The major QTL on chromosome 12, named Pup1 for P uptake 1, controls most of
the variation in P-deficiency tolerance in the Nipponbare/Kasalath population.
Pup1 substantially increased P uptake from P-deficient soils but has no apparent
effect when P is not limiting. Transferring Pup1 to intolerant genotypes increased

P uptake, plant biomass, and grain yield by over threefold on a P-fixing soil
(Wissuwa and Ae 2001b). Near isogenic lines containing Pup1 maintained rela-
tively higher root growth and root surface area in P-deficient soils than their
counterparts lacking the Pup1 introgression. Carbohydrate supply from leaves
to roots did not explain the reduction in root growth in lines missing the
Pup1 introgression under P deficiency as root starch concentration increased in
P-deficient roots (Wissuwa 2005). However, model simulations suggested that only
small changes in root growth are necessary to account for the large effects of Pup1
in enhancing P uptake from P-deficient soils, and these differences were mainly due
to variation in root external uptake efficiency (Wissuwa 2003, 2005). These studies
suggest that the genes involved are probably expressed in root tissue where they
either lead to higher root growth per unit P or improve P uptake per unit root size or
surface area.
Pup1 was recently fine-mapped to the long arm of chromosome 12 within the
physical interval of 15.31–15.47 Mb (Heuer et al. 2009). The genes in this locus
were initially annotated based on Nipponbare reference genome sequence; how-
ever, this annotation did not unveil obvious candidates for P-uptake efficiency.
Subsequently, the locus was sequenced in the original donor parent Kasalath, and
this revealed significant variation with the reference sequences of both Nipponbare
and 93-11, with considerable distinction in size differences caused by insertions
and deletions, together with a large number of transposon and retrotransposon-
related sequences (Heuer et al. 2009). This variation highlighted the significance
of sequencing QTL regions in the donor parent targeted for cloning, as the
underlying genes might be lacking in the two reference genomes that are currently
available. Similar observations were also made when cloning the SUB1 gene
associated with tolerance of submergence in rice (Xu et al. 2006). Several of
the newly annotated genes using Kasalath sequence are novel and are mainly
located within the insertion–deletion regions. Detailed analysis of these genes
annotated from the Kasalath sequence is ongoing and their potential role in
tolerance of P deficiency is being depicted based on physiological evidence and
sequence analyses. Identifying and cloning of Pup1 will help in designing precise
gene-based markers for use in breeding and for revealing its physiological and
molecular bases, particularly its effects on root growth under P-deficiency. This
information could also be important for enhancing tolerance in other crop species
by identifying Pup1 homologs.
A marker-assisted breeding system to introgress Pup1 into popular varieties is
also being developed, and its contribution for enhancing tolerance of P deficiency in
a wider range of genetic backgrounds and under natural field conditions is being
further quantified. SSR markers linked to the Pup1 locus were identified and tested
in a few accessions, and some of them were found to be specific to Kasalath donor
parent alleles, suggesting their potential use for monitoring the Pup1 introgression
during backcrossing (Collard et al. 2006). PCR-based markers were also developed
based on the genes annotated at the Pup1 locus and are currently being used to
transfer Pup1 into a few upland and lowland popular varieties using MABC,
following the strategy used for SUB1 locus (Septiningsih et al. 2009). Cloning of
the gene responsible for Pup1 action will accelerate the development of this marker
system. Combining Pup1 with qREP-6 into the background of popular varieties and
advanced breeding lines could significantly enhance their performance under
P-deficient soil conditions.
12.2.2.2 Zinc Deficiency
Zinc deficiency is a widespread soil constraint for rice production, with about 50%
of lowland rice soils believed to be Zn-deficient. Zn deficiency can result from low
total soil–Zn content, but it is more frequently caused by Zn immobilization in the
soil. A range of soil conditions have been associated with binding it in forms that
are less readily available for plants, such as alkaline pH, prolonged submergence
and low redox potential, high organic matter and bicarbonate content, high Mg:Ca
ratio, and high available P (Yoshida et al. 1973; Forno et al. 1975; Neue and Lantin
1994; White and Zasoski 1999). High soil pH and bicarbonate appear to be the main
factors associated with the widespread Zn deficiency in calcareous soils as the case
of the Indo-Gangetic Plains of India and Pakistan (Qadar 2002), whereas perennial
wetness and low redox potential are the major causes of Zn deficiency in peat and
coastal saline soils (Neue and Lantin 1994; Quijano-Guerta et al. 2002). Similar to
P solubilization under flooded soils, rice roots can solubilize Zn through acidifica-
tion of the rhizosphere in the vicinity of the roots (Kirk and Bajita 1995) through the
release of H+ from the roots or during oxidation of iron by O2 released from roots.
Zinc deficiency can be effectively eliminated by using Zn fertilizers; however,
the high cost associated with applying sufficient Zn places a considerable burden on
farmers, particularly in rainfed areas of Asia, where most soils demand high Zn
application as a consequence of its immobilization in the soil. Breeding efforts to
develop rice cultivars that are more efficient in Zn uptake from these soils should
therefore be intensified to improve tolerance of Zn deficiency in rice (Quijano-Guerta
et al. 2002; Ismail et al. 2007). Incorporating tolerance of Zn deficiency also seems to
improve performance under other abiotic stresses such as alkaline soils, salinity,
P deficiency, and peat soils (Singh et al. 2004; Quijano-Guerta and Kirk 2002;
Quijano-Guerta et al. 2002). However, the mechanisms of this cross-tolerance still
awaits further investigation and may be attributed solely to better Zn acquisition when
Zn is most limiting, with the consequent improvements in root health and growth.
The major mechanisms associated with Zn deficiency tolerance in plants are still
poorly understood and several mechanisms were suggested (Hacisalihoglu and
Kochian 2003); however, the effectiveness of these different traits as well as their
physiological and molecular bases are still incomplete. Multiple symptoms are
generally observed in rice in Zn-deficient soils, including development of brown
spots on leaves that eventually entirely cover older leaves (leaf bronzing), stunted
plant growth and poor root development, and seedling mortality in severe condi-
tions. Flowering is normally delayed or even hindered and grain yield substantially
decreases (Ismail et al. 2007). These symptoms are largely under independent
genetic control as different QTLs were associated with traits such as leaf bronzing
and plant mortality (Wissuwa et al. 2006). The results largely suggest multiple
tolerance mechanisms that can either operate in root or shoot. Mechanisms asso-
ciated with Zn uptake and root growth obviously reside in roots, whereas mechan-
isms associated with reduced leaf bronzing likely occur within leaf tissue. Our
recent studies suggested that tolerance to Zn deficiency in flooded Zn-deficient soils
was associated with rhizosphere processes that enhance availability and uptake
of Zn rather than with shoot traits or internal efficiency (Wissuwa et al. 2006).
Effects of Zn Deficiency on Root Growth in Rice
Zinc uptake into roots is either as Zn2+ ion or as a Zn-phytosiderophore complex, and
as for most cations, its transport is mediated by a low-affinity transport system and a
high-affinity system, with the latter dominating under Zn deficiency (Hacisalihoglu
et al. 2001). However, the molecular nature of these systems remains poorly under-
stood. In conditions when Zn availability is low due to binding of Zn in the soil,
adaptive root mechanisms that increase Zn availability through desorption of Zn from
binding sites in the soil are likely more important than transmembrane transport
systems. Release of Zn from soil-bound forms has been linked with two classes of
compounds secreted from plant roots, phytosiderophores, and nonprotein amino acids
that chelate a number of micronutrients (Rengel et al. 1998; Suzuki et al. 2006) and
organic acids such as citrate and malate, which were also thought to be involved in
both Zn and P deficiency tolerance in rice. The involvement of a rhizosphere effect in
maintaining Zn uptake under field conditions was further supported by the observa-
tion that increasing the plant density per hill increased shoot dry matter and Zn uptake,
with no apparent symptoms of Zn deficiency (Hoffland et al. 2006).
Root growth in rice is severely inhibited under Zn deficiency, and tolerant
genotypes tend to maintain their ability to regenerate new roots and maintain higher
root biomass in Zn-deficient soils. In both calcareous and heavily submerged soils,
Zn deficiency typically coincides with high bicarbonate concentration in the soil
solution, and sensitive genotypes showed strong suppression in root growth in
response to bicarbonate, with consequent reduction in Zn acquisition. The negative
effect of bicarbonate is probably caused by excess accumulation of organic acids
within the roots of sensitive cultivars, whereas tolerant genotypes avoid this effect
by maintaining higher rates of organic acid excretion. This might also help in
mobilizing Zn in soil solution and enhance its accessibility by plant roots, resulting
in further root growth in tolerant genotypes, commonly seen as early as 2 weeks
after transplanting in Zn-deficient soils (Hajiboland et al. 2005; Ismail et al. 2007).
Germplasm Improvement for Zn Efficiency Tolerance
Genetic variability in the ability to grow under low Zn conditions has been observed
in rice (Quijano-Guerta et al. 2002; Yang et al. 1994). However, despite this genetic
variability and the dire need to develop Zn-efficient varieties, no formal breeding
program has yet been initiated to develop more Zn-efficient varieties. Limited
progress was achieved indirectly when selecting for tolerance of other soil pro-
blems as in alkaline soils of north India (Singh et al. 2004). Our recent efforts aimed
to identify genotypes contrasting in their tolerance of Zn deficiency under natural
field conditions to understand the mechanisms of tolerance and to develop strate-
gies to incorporate tolerance through breeding.
Identification of QTLs with reasonably large effects on Zn deficiency tolerance
is a crucial first step that will allow the eventual incorporation through MABC as
well as the identification of tolerance genes after further fine-mapping and
subsequent positional cloning. Using a mapping population developed from the
indica genotype IR74 (sensitive) and Jalmagna (tolerant), several QTLs associated
with plant mortality, leaf bronzing, and biomass were detected on a Zn-deficient
field, with only one minor QTL for plant mortality colocalized with a QTL for leaf
bronzing (Wissuwa et al. 2006). QTLs for plant mortality acted in a purely additive
manner, whereas digenic interactions were important for leaf bronzing and for
shoot biomass, and in both cases, the epistatic interactions involved the main
QTL for plant mortality mapped on chromosome 12. Currently, several of these
QTLs are being targeted for fine-mapping for further genetic dissection and for use
in breeding. Advancing our knowledge of the mechanisms of tolerance together
with the identification of genes responsible for the mapped QTL regions will enable
a precise MABC strategy to speed up breeding for tolerance of Zn deficiency.
12.2.3 Mineral Toxicity
Approximately 30% of the earth’s lands are classified as acidic and about half of the
potentially arable land is acidic (von Uexkull and Mutert 1995). Soil acidity limits
crop production through a combination of nutrient toxicities and deficiencies. These
soils constitute a serious constraint across vast portions of rice-growing areas of the
tropics. Besides mostly being deficient in major plant nutrients such as P, they also
contain toxic concentrations of other elements such as aluminum and iron, as both
Al3+ and Fe2+ ions become soluble under low pH. These in turn damage the root
system, and their excessive uptake leads to toxicity within the plant, leading to
decreased growth and yield. Research on the genetic control of tolerance of the
stresses encountered in acid soils in rice is still in its early stages despite their
enormous effects on rice production in affected areas.
12.2.3.1 Aluminum Toxicity
Aluminum is the most abundant metal in the earth’s crust, constituting approxi-
mately 7% of the soil and is predominately found in clays. Under low pH (<5), it is
solubilized as Al3+ in soil solutions, which is highly toxic to plants. Aluminum
toxicity is the main factor limiting the productivity of crop plants in acid soils,
particularly in the tropics and subtropics. A high concentration of Al3+ severely
hampers root growth, with consequent inhibition of water and nutrient uptake,
resulting in severe reduction in growth and productivity. Al toxicity has been
extensively studied in several plant species and particularly in grasses, including
wheat, sorghum, maize, and rye (Kochian et al. 2004, 2005). The primary mecha-
nism of tolerance identified in most of these crops involves the exudation of organic
acids from the root apex, which in turn binds aluminum and excludes it from
entering the root, as was first identified in wheat (Delhaize et al. 1993). Several
organic acid exudates were documented in several plant species such as malate
exudation in wheat and Arabidopsis, citrate exudation in maize, sorghum, and
soybean, and both citrate and malate in rye, Triticale, and oilseed rape (Kochian
et al. 2004). Another potential mechanism involves tolerance of high Al accumula-
tion in roots and shoots tissue through internal detoxification (Ma et al. 1998).
Recently, genes that control tolerance of Al toxicity were cloned from wheat and
sorghum (Sasaki et al. 2004; Magalhaes et al. 2007), and in both crops, tolerance of
Al toxicity was attributed to the exudation of organic acids by roots to serve as
chelates and detoxify Al3+ in the rhizosphere, particularly around the actively
growing root tips, which are the main site of Al toxicity.
Aluminum toxicity is a major limitation to rice production in both rainfed
lowland and upland soils. Rice is the most tolerant cereal; however, little is
known regarding the physiology of this tolerance. Mechanisms of tolerance in
rice are expected to act differently compared with other cereals due to the low
organic acid excretion by rice roots, which is unlikely to play a major role in Al
detoxification in the rhizosphere. A few reports have suggested exclusion of excess
Al at the root tip to be involved in rice tolerance of Al toxicity; however, these
studies were limited to only two genotypes, one tolerant and one sensitive (Ma et al.
2002; Yang et al. 2008). Apparently, novel mechanisms are probably involved in
the high levels of Al toxicity tolerance in rice. Understanding these mechanisms
and the gene(s) underlying the tolerance traits will facilitate further improvement of
rice varieties and development of varieties of other cereals with higher tolerance of
Al toxicity than the existing varieties.
Numerous studies have identified QTLs associated with Al toxicity tolerance in
rice (Table 12.1). For example, Wu et al. (2000) identified several QTLs associated
with Al tolerance in a recombinant inbred mapping population derived from
Azucena and IR1552. Nguyen et al. (2001) also detected five QTLs for Al toxicity
tolerance distributed on five chromosomes, with a major QTL located on chromo-
some 1. Using a double haploid population developed from CT9993 (tolerant) and
IR62266 (sensitive), Nguyen et al (2002) identified 20 QTLs controlling root
growth under Al toxicity stress and control conditions, distributed over ten chromo-
somes, with the two largest QTLs identified on Chromosomes 1 and 8. The region
on chromosome 1 was found to be conserved across several genetic backgrounds,
and therefore, could be targeted for use in breeding as well as for subsequent
cloning. Using a backcross population derived from Koshihikari (tolerant) and
Kasalath (intolerant), Ma et al. (2002) identified three QTLs on chromosomes 1, 2,

and 6, collectively explaining about 27% of the phenotypic variability in Al toxicity
tolerance in this population. In an RIL population derived from the cross of Oryza
sativa (IR64, sensitive) and Oryza rufipogon (tolerant), three QTLs were identified
for root length under Al toxicity stress and five for relative root length. O. rufipogon
contributed all favorable alleles for each of the five QTLs for relative root length as
the most important trait affected by Al toxicity. Individually, these QTLs explained
9–25% of the phenotypic variation. The QTLs for relative root length on chromo-
somes 1 and 9 were observed to be consistent among different rice populations. The
major QTL explaining 25% of the phenotypic variation was on chromosome 3 of
rice, and was conserved across cereals, suggesting the potential for its use in
breeding (Nguyen et al. 2003). Recently, Xue et al. (2006) identified three QTLs
on chromosomes 1, 9, and 11, using an RIL population derived from a japonica
cultivar Asominori (tolerant) and an indica cultivar IR24 (sensitive), with pheno-
typic variance of 13–18%; the two QTLs on chromosome 1 and 9 also were found to
be consistent among different rice populations. In a subsequent study, the QTL on
chromosome 9 was fine-mapped using a high-resolution physical map, and linked
markers that cosegregated with this QTL were identified (Xue et al. 2007). These
studies indicated the complexity of Al toxicity tolerance in rice; however, identifi-
cation of similar QTLs across different populations and backgrounds suggested that
these QTLs could be targeted for breeding through MABC. Subsequent studies are
also needed to advance our knowledge beyond the identification of QTL loci.
12.2.3.2 Iron Toxicity
Iron toxicity is a nutrient disorder, caused by excessive uptake of ferrous ions in

amounts that disrupts metabolic processes, resulting in injury and reduced growth
and yield. It commonly occurs in highly reduced soils when toxic concentrations of
ferrous iron accumulate in soil solution, or when inflow carries soluble iron from
upper slopes into highly reduced low lying areas. It is also a common problem in
acid sulfate rice soils, as in Vietnam, Thailand, Bangladesh, and Indonesia. In West
Africa, iron toxicity is widespread throughout the humid tropics, affecting about
30–40% of the cultivated lowlands.
Iron Toxicity in Rice and Bases of Tolerance
Iron toxicity was first reported in rice by Ponnamperuma et al. (1955) when they
attributed the bronzing disease of lowland rice to high concentration of ferrous iron
in soil solution and its subsequent excessive uptake and accumulation in plant
tissues. Since then, iron toxicity has been recognized as one of the most widely
spread micronutrient disorders, especially in the humid tropics of Asia, West and
Central Africa, and South America, particularly in acid, acid sulfate, and peat soils
(Dobermann and Fairhurst 2000; Balasubramanian et al. 2007; Fageria et al. 2008).
Large areas of these wetlands ideally suited for rice production remain underused or
even unused in severe cases. In West Africa, yield losses of 12–100% were reported,
depending on the severity of the stress and the extent of tolerance of the varieties being
grown. Symptoms of damage are expressed as rusty leaf spots (bronzing), stained leaf
edges, and dark-brown rigid and poorly developed roots. The typical visual symptom
in rice is the bronzing of leaves, and the yield losses associated with the appearance of
these bronzing symptoms commonly range from 15 to 30%; however, severe stress
can cause complete crop failure (Audebert and Sahrawat 2000).
The physiological basis of tolerance of iron toxicity in rice has been studied by
various investigators, and a few strategies were proposed (1) exclusion of ferrous
irons by roots through root selectivity to avoid excessive uptake; (2) proper
compartmentation through apoplastic immobilization or storage in less active
tissues such as older leaves, leaf sheaths, old roots, and stems; and (3) high tissue
tolerance, probably through sequestration in vacuoles or enzymatic detoxification
in the symplast. Formation of iron plaque on the root surface in soils containing
high concentrations of ferrous iron in solution could also be another strategy to
reduce its uptake. Plaque formation is caused by oxidation of ferrous irons by
oxygen that leaks from rice roots to form the insoluble ferric irons, which then
precipitate on the root surface. Presumably, several mechanisms could be involved
in enhancing tolerance of rice to iron toxicity; however, the genetic and molecular
bases of these mechanisms are still not well understood.
Germplasm Improvement for Iron Toxicity Tolerance
Substantial genetic variation has been reported in rice in response to high ferrous
iron concentration in soils or in hydroponics (Gunawardena et al. 1982; Sahrawat
et al. 1996; Fageria et al. 2008). This makes it possible to breed rice cultivars with
greater tolerance of iron toxicity, which could substantially enhance rice production
in affected areas. However, despite this genetic variability, still little progress was
made in developing tolerant varieties that are high-yielding. Several studies have
identified QTLs associated with tolerance in rice. Wu et al. (1997) identified three
QTLs, two of them on chromosome 1 and one on chromosome 8, using a mapping
population derived from the tolerant japonica Azucena and the moderately sensi-
tive indica variety IR64. The phenotypic contribution of these QTLs ranges from 10
to 32%. Using a backcross population developed from Nipponbare and Kasalath,
Wan et al. (2003) identified four QTLs for various traits associated with Fe toxicity
tolerance, three of them were on chromosome 1, and one on chromosome 3. These
QTLs has LOD scores between 3.17 and 7.03, and phenotypic effects ranging from
20 to 48%. Thus QTLs of major effects on Fe(II) toxicity tolerance are present in
rice and provide future targets for MABC to introgress them into popular varieties
and breeding lines for use in target areas.
12.3 Current and Future Prospects of Marker Assisted

Backcrossing for Breeding Varieties Adapted
to Problem Soils
Genetic linkage maps have made it possible to study the chromosomal locations of
genes for improving yield and other complex agronomic and adaptive traits impor-
tant in agriculture (Tanksley and McCouch 1997). Genetic mapping studies have
led to over 8,500 QTLs identified for many different traits in rice, including
tolerance to abiotic stresses (www.gramene.org). At the same time, advances in
physiology and genomics have led to a more detailed insight into the responses of
rice to soil stresses. While many previous studies explored differential gene expres-
sion between stress and control conditions through microarrays and RT–PCR
(Walia et al. 2005, 2007), a deeper understanding of plant responses to abiotic
stresses is now being investigated through proteomic and metabolomic profiling
(Bohnert et al. 2006; Torabi et al. 2008) and by studying small RNAs (Sunkar et al.
2007). Future techniques in high throughput sequencing will only make these
studies faster and more powerful (Sunkar et al. 2008). By integrating genomic
methods to study key traits with genetic tools such as NIL development and QTL
cloning, a better understanding of key tolerance mechanisms will lead to more
efficient methods for breeding more tolerant varieties (Varshney et al. 2005; Salvi
and Tuberosa 2005). For example, important QTLs and loci identified through
association mapping for different traits can now be combined through marker-
assisted breeding for crop improvement (Takeda and Matsuoka 2008). Furthermore,
as more genomic sequence and SNP data becomes available through resequencing
(McNally et al. 2006) and de novo whole genome sequencing, the genetic variation
of tolerance can be investigated on a scale never before possible. Having more
genome sequence data will be important when dealing with indica varieties as the
tolerant donors, since the gene content between indica and the japonica Nipponbare
reference sequence can be significantly different, as was shown by the recent study
at the Pup1 locus (Heuer et al. 2009). Moreover, high-density SNP arrays will lead to
more powerful association genetic studies that will help explore the useful genetic
variation that is captured in rice germplasm collections. High throughput SNP
genotyping platforms will also enable more efficient MABC by reducing the cost
per marker and by speeding up the process through multiplexing. As more SNP
markers in rice are characterized, then subsets of SNPs that are optimized for
different breeding applications can be selected. For example, a small number of
targeted SNPs at gene loci, including functional SNPs and key SNP haplotypes, can
be used for foreground selection in breeding programs for traits where the QTLs
have already been cloned. In addition, QTL mapping and background selection can
employ low-cost multiplexed sets of 384 SNPs, while QTL fine-mapping and more
precise tracking of introgressions may require larger multiplexed sets of 1,536 SNPs
or even 10,000 SNP chip platforms that are becoming available. By offering rapid,
cost-effective, and robust genotyping, these new technologies will allow the wider
use of the valuable QTLs that have already been identified, and will ultimately bring
marker-assisted selection into mainstream breeding efforts.
12.4 Conclusions
The stresses encountered in problem soils are generally complex, where several
abiotic factors are commonly encountered. This complexity coupled with the
multitude of traits required for plants to withstand a particular stress has made
improvement through conventional breeding a challenging undertaking, as wit-
nessed by the slow progress in previous efforts. New approaches are therefore
necessary to identify the suite of adaptive traits and mechanisms of tolerance,
followed by swift incorporation into varieties and elite material that lack these
traits but meet farmers’ expectations. Considerable progress was made in under-
standing signaling and response pathways for most of the major soil-related pro-
blems, and the recent developments in genomics have provided powerful tools for
genetic dissection of these traits. Despite the complexity of most soil problems,
tolerance of some of them was attributed to a few QTLs of large effects (Table 12.1),
and the recent developments in marker technologies made it possible to tag and
incorporate these major QTLs into high-yielding varieties. Preliminary efforts to
incorporate some of these QTLs have demonstrated measurable effects on the
performance of rice varieties under stress. The recent developments in high
throughput genotyping systems also hold great potential in overcoming the obsta-
cles encountered in MABC. Complementing conventional methods with MABC
will continue to help accelerate the development of more resilient varieties that
could positively impact productivity of rice on problem soils.
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Index
A Alt4, 271, 273, 274, 276, 277, 281, 283, 289

ABA. See Abscisic acid Alt gene, 271
ABA1 gene, 51, 56, 62 Al toxicity, 21
Abiotic and biotic stresses, 15, 16 AltSB, 150, 154–156, 275, 277, 282, 284, 291
Abiotic stress Aluminum
cold, 248, 258 tolerance mechanisms, 316
drought, 248, 258 toxicity, 315–317
heat, 248 Amplified fragment length polymorphisms
tolerance, 18–19 (AFLP), 272, 287, 290
Abscisic acid (ABA), 132, 134, 137, 194, 196, Appressorium, 94, 96
203–205 Aquaporins, 224, 225
Acid-soil-resistant, 272 Arabidopsis, 128–130, 132, 136–140, 277, 279,
Acid soils, 266–268, 270, 272, 273, 287, 292 281–283, 285–287
Adaptive response of crops to drought and low Arabidopsis thaliana, 20, 25, 43, 72–74, 151
nutrients, 194 natural variation and mutagenesis, 115–118
Additiveadditive gene interaction, 255 root exudates, 116, 117
Additive gene effects Arbuscule, 88, 93, 95, 96, 100, 101
Adventitious roots, 17, 19 Argentina, 148
Adventitious root system, 26 Association mapping, 273, 289, 290
Aegilops strigosa, 274 AtALF5, 282
Aegilops tauschii, 272, 291 AtALMT1, 152, 153
Aegilops uniaristata, 270, 291 AtFRD3, 283, 287
Al accumulation, 266, 268 ATP-binding cassette (ABC) transporter,
Al-activated citrate transporter, 282, 286 123, 285
Al-activated efflux, 281 Autoregulation, 101, 102
Al-activated exudation, 278, 281 Auxin, 16, 17, 30, 37, 39–42, 44–47, 50–58, 94,
Al-activated malate efflux, 279, 281, 97, 98, 101, 129–133, 135, 137, 139
283, 286 Auxin-responsive protein/indole acetic acid
Al-activated malate transporter, 281, 286 induced protein repressor (AUX/IAA
Al-activated organic acid efflux, 278, 279 repressor), 223
Alanine aminotransferase (ALT), 19
Alfalfa, 286 B
Al-induced malate release, 271 Bacteria, 87, 88, 92, 93, 95, 97, 99, 102
Allele mining, 290–291 Bacteroids, 99, 100
Alm2, 274 Barley, 25–74, 266, 267, 270, 273–288,
ALMT1, 276, 279, 283–285 290, 291
Alp, 274, 276, 277 Bioinformatics approach, 72
Alt3, 271, 273, 274, 289 Bioinformatic tools, 22

DOI 10.1007/978-3-540-85546-0, # Springer-Verlag Berlin Heidelberg 2011
314 Index
Biotic stress Defense responses, 88, 89, 95–97, 101

Ascochyta blight, 248 Development, 88–90, 94, 95, 97–100, 102,
Fusarium wilt, 248 127–139
tolerance, 18 Ditelosomic chromosome stocks, 150
BLASTn and BLASTp, 60, 73 DNA sequencing procedures, 66
BODENLOS (BDL) mutants, 38, 55 Dominant loci, 271, 273
Brachiaria decumbens, 157, 159 Downregulated transcripts, 282
Brassica, 281 Downstream sequences, 280, 284
Brassica napus (rapeseed), 153, 281 Drought, 128, 135–137
Brassica oleracea, 281 Drought escape, 249
Brazil, 148 Drought-tolerant index, 251
Bronzing, 170, 172, 173, 180 Dry bean, 247
C E
Camellia sinesis (tea), 159 Endoparasitic nematodes, 88, 97, 98
Candidate gene, 203, 205–206, 208, 258–260, Environment, 127–140
285–286, 290 Environmental constraints, 135
Candidate gene markers, 272 Epidermis cells, 19
Canola, 286, 287 Epistatic effects, 274, 289
CAP3 program, 66 Eriochrome cyanine, 268, 288
CAPS marker, 280 Ethylene, 132, 136, 137
“Caspian strip,” 36 Exon/intron arrangement, 66, 67
Cation, 172 Expressed-sequence tags (ESTs), 20, 25–74,
cDNA macroarray, 256 256, 257, 259
CEC, 172 External resistance mechanisms, 278
Cell cycle, 97, 98, 129, 130, 132, 133
Cell division, 89, 97, 98 F
Cercozoa, 121 Fagopyrum esculentum (buckwheat),
Chelation, 177 156, 159
Chickpea, 247–260 Field pea, 247, 254
Cicer arietinum, 237 Flavonoids, 92
Citrate, 148, 153–156, 159, 278, 279, 281–284, Flooding, 200–201
286, 287 Flooding conditions, 200–201
efflux, 277, 279, 283–286 “Founder cells,” 55
synthase, 285, 286 FRD3, 156
CLE peptide, 98 Fungi, 87, 88, 91–96, 98–102
COBRA-like protein, 60
Colinearity breakage, 276 G
Colocasia escultenta (taro), 156 GenBank databases, 60, 61, 66
Colombia, 148 Gene-based functional molecular markers
Comparative mapping, 275–277 EST-SNPs, 256
Core-break technique, 230 EST-SSRs, 256
Crack-entry, 88, 95 single feature polymorphisms, 256
Crop productivity Gene-based marker, 270, 290
nutrient, 225–227 Gene effects, 255
root, 219–221 Genes, 177–181, 186
root growth, 221–224 Genes controlling root system, 26, 27
water and nutrient uptake, 224–225 Genesis, 181–183
Cytokinin, 97, 98, 132, 134, 135, 137 Genetic approaches, 26
Cytokinin receptor, 36 Genome
assembly, 257
D sequencing, 89–91, 93, 102
DArT, 272, 287, 290 Genome-specific markers, 270
Data-mining, 60–72 Genomic resources
Index 315
BAC and BIBAC libraries, 259 L

BAC-end sequences, 259 LaMATE, 283
ESTs, 259 Laser capture micro-dissection, 99
expression genetics, 259 Lateral root (LR), 34, 36, 37, 39, 45–53, 55, 56,
genetical genomics, 259 61, 68, 71, 127–139
solexa tags, 259 Lateral root formation, 92, 97, 98
SSR markers, 259 Legume, 127, 128, 132–137
Giant cells, 89, 99 Locus XME, 271
Gibberellins (GAs), 134 Lodging conditions, 198, 201–202
GL2 mutant, 27 Long distance communication, 100
Glycine max (soybean), 72, 153 Lotus japonicus, 226
GNOM1, 17 Low nitrogen conditions, 198–199, 208
Grain yield (GY), 194–199, 203–205 Low nutrients, 194, 198
Low phosphorus conditions, 199–200
H Low temperature, 198
Harvest index (HI), 251 Lupin, 281, 283
Helix-loop-helix transcription factors, 17 Lupinus albus, 283
Hematoxylin, 268, 269, 288, 290
Hexoquinase (HXK), 19 M
High pressure liquid chromatography Macrosynteny, 276, 277
(HPLC), 115 Maize, 266–268, 270, 274–277, 281, 289
High-resolution mapping, 275, 277, Maize roots, 193–209
282, 283 Major genes
Homogeneity and heterogeneity, 17 efl-1, 249
Hordeum vulgare (Barley), 151, 154–156 ppd, 249
HvAACT1, 279, 282, 284, 286 QTL effects, 289
HvALMT1, 276, 281 Malate, 148–157, 271, 278, 279, 281, 284,
HvMATE, 155, 156, 276, 277, 283–286, 286, 287
288, 290 Malate efflux, 270, 271, 279, 281, 283, 286
HvMATE/HvAACT1, 279 Map-based cloning, 275
HXK. See Hexoquinase Marker-assisted back crossing (MABC), 22
Hydrangea, 159 Marker-assisted selection (MAS), 250, 258,
Hydroponics, 230, 268 267, 275, 287–290
Hydrotropism, 40, 55 MATE. See Multidrug and toxic compound
Hydroxyamates, 160 extrusion
Hypernodulation, 98 Medicago (M. trucatula), 128, 132, 133,
135–137, 139, 236
I Medicago sativa (alfalfa), 153, 156
Incremental crop tolerance (ICT), 269 Membrane-localized transporter, 279
InDels, 280, 290 Meristem, 127, 128, 130–134, 139
Infection threads (IT), 95 Metabolism, 176, 178, 181, 182
Inhibition of root growth, 267 Metabolite profiling, 90
Internal resistance mechanisms, 278 Metagenomic enrichment technology, 119
International Medicago Genome Annotation Microarray, 90, 98, 131, 136
Group (IMGAG), 257 MicroRNA-mediated signaling, 17
Introgression lines (ILs), 203 MicroRNAs (miRNAs), 135, 138–139
Introns, 277, 280, 283, 284, 290 Millet, 266
Iron Model legumes, 89–91
tolerance, 318 Molecular and hormonal regulation, 221–224
toxicity, 317–318 Molecular breeding, 16, 19–21, 265–292
marker-assisted backcrossing (MABC),
J 307, 319
Jellyfish program, 66, 73 SNP genotyping, 319
316 Index
Molecular breeding (cont.) limited mobility in soil, 309

SSR markers, 312 tolerance mechanisms, 310–312
Molecular markers, 250, 255, 256, 258–260, uptake, 310–313
272, 275, 287, 291 Pht locus, 274
Monocots and dicot, 17 Phylogenetic analysis, 67–68
Monocotyledonous species, 26, 55–60, 66–67, Physcometrilla patens, 72
69, 72 Phytohormones, 18, 137
MONOPTEROS (MP) mutants, 38, 55 Plant genetic determination, 119–122
Morphogenesis, 130, 131 Plant-soil-water interactions, 15
Multidrug and toxic compound extrusion Plasma membrane, 266, 281, 284, 285
(MATE), 277, 279, 282–285, 287 Plasticity, 128, 132, 135–139
Multigenic inheritance, 270 Pleiotropic effect, 27
Mutagenesis, 91 Polymorphic content (PIC) value, 290
Mutant traits, 25–74 Positional cloning, 203–205, 209
Mu transposon, 56 Potential orthologs, 66–67
Myc factor, 93, 94 Problem soils
Mycorrhizal fungi, 88, 92, 96, 98, 100–102 mineral deficiency, 309–315
mineral toxicity, 315
N Promoter region upstream of TaALMT1, 284
Near isogenic lines (NILs), 196, 202–206 Proteomics, 90, 95, 96, 98, 99
Nematodes, 87–91, 93, 97–100, 102 Proteomics and metabolomics, 18
Nicotiana, 154 Protoxylem, 34, 36
Nitroblue tetrazolium (NBT), 268 Pulse chip, 258
Nitrogen-fixing bacteria, 88, 99 PVC cylinder culture system, 253
Nod factor receptor, 88, 93, 98
Nod factors, 88, 92–95, 97, 98 Q
Nodule, 88, 90, 93–95, 97–101, 127, 128, Quantitative trait loci (QTL), 16, 19–21, 150,
132–135, 139 151, 162, 195–205, 208, 209, 255,
Nutrient exchange, 88, 89, 99–100 257–260, 271, 272, 274, 275, 277, 281,
Nutrient solution culture, 267–269 287, 289
aluminum toxicity tolerance, 308
O fine-mapping, 307–309, 315, 319
Oat, 266, 267, 270, 274–276, 278, 289 iron toxicity tolerance, 308, 318
Oomycetes, 88, 90, 91 P-deficiency tolerance, 308, 309,
Organic acids, 310, 314, 316 311, 312
Organic acid transporters, 279, 283 Pup1, 311–313
Organogenesis, 129, 130, 133, 134 salinity tolerance, 307–309
Oryza sativa (rice), 149, 157 Saltol, 309
Osmotic adjustment, 253 Zn deficiency tolerance, 308
Oxalacetate, 278 “Quorum sensing signals,” 92
Oxalate, 156, 158, 159
Oxidative stress, 285 R
Oxygen Isotope Ratio, 232–233 RAPD, 272, 290
Reactive oxygen species, 148, 160
P Reduction, 170, 171, 174–177, 180, 183, 184
Panicum virgatum, 122 Regulatory genes, 137, 140
Pea, 266 Relative root growth, 268, 289
pH, 171, 172, 176, 178, 184 Reverse genetic approaches, 91, 102
pH buffering, 158 Reversible phosphorylation, 150–152
Phenolics, 157, 159, 160 RFLP, 272, 287, 289, 290
Phosphate, 152, 157 Rhizobia, 88, 90–95, 97–99, 101, 102
Phosphorous Rhizobium, 127, 133, 134
deficiency, 309–313 Rhizosphere, 87, 92, 93
Index 317
Ribulose 5 phosphate 3 epimerase root proliferation, 250

(R5P3E), 276 transgenic approach, 236–237
Rice, 266–268, 270, 273–280, 282, 287, R5P3E. See Ribulose 5 phosphate 3 epimerase
289–291 Rye, 266–271, 273–281, 283, 289
improvement for iron toxicity, 318
improvement for P-uptake, 311–313 S
improvement for salinity tolerance, Salinity (salt), 128, 135–138
306–309 HKT transporters, 307
improvement for Zn efficiency, 314–315 physiological mechanisms, 306
sequence variation, 312 Pokkali, 307–309
Rice genes, 66 tolerance in rice, 306, 307
RNA interference (RNAi), 91, 92, 97 SbMATE, 279, 284, 286, 290
Root ScALMT1, 276, 277, 281, 283, 289
apex, 266, 271, 283 ScMATE, 276, 277
architecture, 25–74, 127–139, 193–209 Secale cereale (rye), 153, 156
biomass, 252, 253, 255 Semi-arid tropics, 247–249
characteristics, 25 Seminal root system, 26
citrate exudation, 282, 284 Senescence, 96, 100
development, 16–18, 21 Shoot-borne root system, 26
elongation, 266–268 Shotgun sequencing, 119, 120
genomics, 15–22 Signaling pathways, 102
growth, 16–19, 21, 304–318 Simple sequence repeat (SSR), 272, 276, 277,
growth in hydroponics, 197, 198 287, 289, 290
hairs, 310 SLR1 and SLR2 mutants, 60
hydraulic conductivity, 253, 254 Smith-Waterman algorithm, 61
knot and cyst nematodes, 88, 89, 91, 93, SNP, 277, 280, 281, 287, 290
97–99 Soil
length, 308, 310, 317 acidic, 315
microbe interactions, 20 alkaline, 303, 304, 309, 313, 315
mutants, 26–60 environment, 127, 128, 132, 135–137, 140
plaque formation, 318 microbes, 114, 115, 119, 122
size, 310, 312 rhizobia, 21
traces, 17 Solexa sequencing technology, 257
Root apical meristem (RAM), 16 Sorghum, 266, 267, 270, 275–280, 282, 284,
Root-associated microbes, 119–122 285, 290, 291
Root dry weight (RDW), 254, 255 Sorghum bicolor, 121
Root-external microbes (REM), 119 Sorghum propinquum, 121
Rooting depth (RD), 250, 254 Spatial control, 137–138
Root-internal microbes (RIM), 119 SSR motifs, 280, 290
Root length density (RLD), 250, 252, 254, 255 Stele, 27, 31, 34, 36, 56
Root-microbe interaction, 119 STOP1, 153
Root permeability Stress, 128, 135–140
apoplastic, 253 Supernodulation, 101
symplastic, 254 Suppression subtractive hybridization
transcellular, 254 libraries, 286
Root trait Symbiosis-specific genes, 94
breeding, 234–236 Synctia, 89, 99
drought tolerance, 227–228 Synteny detection, 68–72
genetic variability and crop gowth, Systemic changes, 96
233–234
measurement, 230–232 T
oxygen isotope ratio, 232–233 TaALMT1, 149–152, 154, 270, 272, 276, 277,
root depth, 250 279–281, 283, 284, 286, 288–291
318 Index
TaMATE, 277, 279 V

Tandem repeats, 284, 285 Vacuolar proton transporting
T-DNA insertion, 26–35, 37–53, 57 ATPase, 19
Terminal drought, 248–252, 254 pyrophosphatase, 19
Terminal drought intensity, 252 Vapor pressure deficit (VPD), 253
TILLING populations, 259 Vigna unguiculata (cowpea), 153
Time-dependent effect, 19
Tobacco, 281, 284, 286, 287 W
Toxicity, 170–177, 180–186 Wheat, 266–273, 275–281, 283–288,
Transcriptional changes, 138 290–292
Transcription factors (TF), 131, 136–140, Wild-type chromatograms, 116, 118
221–223
Transcriptome, 205–208 X
Transformation, 279, 286–287 Xenopus, 284
Transgenic roots, 137 Xenopus oocytes, 281, 284, 286
Transgenic technology, 236–237 Xylem groups, 36, 56
Transgenic tobacco, 284, 286
Transpiration (T), 250, 251, 253
Y
Transpiration efficiency (TE), 250, 251
Transport, 175, 177–180, 182, 186 Yield, 194–199, 202–205, 209
Yield stability, 201
Transposon-tagging strategy, 275
Trichoblasts, 27, 28
Triticale, 266, 270, 271, 273–274, 278 Z
Triticum aestivum (wheat), 149–153, 156, Zea mays (maize), 148, 153, 154, 156–161
157, 161 Zinc
Tritticum turgidum, 269 deficiency, 313–315
TTG1 mutants, 27 immobilization in soil, 313
tolerance mechanisms, 314
U uptake into roots, 314
Uptake, 176–179, 181 ZmALMT1, 281

Root Genomics

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Root Genomics

ISBN 978-3-540-85545-3 e-ISBN 978-3-540-85546-0

# Springer-Verlag Berlin Heidelberg 2011

Cover design: deblik Berlin, Germany

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Root Biology: An Inconvenient Truth

With the emerging recognition that agriculture needs to approach sustainability,

Pelotas-RS, Brazil Antonio Costa de Oliveira

1 Introduction to Root Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 EST-Based Approach for Dissecting Root Architecture

3 Genomics of Root–Microbe Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

4 Plant Genetics for Study of the Roles of Root Exudates

5 Impact of the Environment on Root Architecture

6 Mechanisms of Aluminum Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

7 Root Responses to Major Abiotic Stresses in Flooded Soils . . . . . . . . . 155

8 Genomics of Root Architecture and Functions in Maize . . . . . . . . . . . . 179

9 Phenotyping for Root Traits and Their Improvement Through

10 Genomics and Physiological Approaches for Root Trait Breeding

11 Molecular Breeding of Cereals for Aluminum Resistance . . . . . . . . . . 251

12 Molecular Breeding of Rice for Problem Soils . . . . . . . . . . . . . . . . . . . . . . . 289

Ehab Abou-Kheir Department of Crop Physiology, University of Agricultural

Jeffrey L. Bennetzen Department of Biological Sciences, Purdue University,

Srinivasa R. Chaluvadi Department of Genetics, University of Georgia, Athens,

Sergio Conti Department of Agroenvironmental Sciences and Technology, Viale

Martin Crespi Institut des Sciences du Végétal, C.N.R.S, 91198 Gif-sur-Yvette,

Laura de Lorenzo Institut des Sciences du Végétal, C.N.R.S, 91198 Gif-sur-

Axel de Zélicourt Institut des Sciences du Végétal, C.N.R.S, 91198 Gif-sur-

Aparna Deshpande Department of Biological Sciences, Purdue University, West

Anouck Diet Institut des Sciences du Végétal, C.N.R.S, 91198 Gif-sur-Yvette,

Elisabetta Frascaroli Department of Agroenvironmental Sciences and

Silvia Giuliani Department of Agroenvironmental Sciences and Technology,

Véronique Gruber Institut des Sciences du Végétal, C.N.R.S, 91198 Gif-sur-

Perry Gustafson USDA-ARS, University of Missouri, 206 Curtis Hall, Columbia,

Owen A. Hoekenga US Department of Agriculture, Robert W. Holley Center for

Abdelbagi M. Ismail International Rice Research Institute (IRRI), DAPO 7777,

Junichi Kashiwagi Graduate School of Agriculture, Hokkaido University, Kita 9

L. Krishnamurthy International Crops Research Institute for the Semi-Arid

Pierangelo Landi Department of Agroenvironmental Sciences and Technology,

Aleksander Ligeza Department of Genetics, Silesian University, Katowice,

Fang Lu Department of Genetics, University of Georgia, Athens, GA 30602, USA

Jurandir V. Magalhaes Embrapa Maize and Sorghum, Rod. MG 424 Km 65,

Miroslaw Maluszynski Department of Genetics, Silesian University, Katowice,

Ulrike Mathesius Australian Research Council Centre of Excellence for Integra-

B. Mohanraju Department of Crop Physiology, University of Agricultural

Karaba N. Nataraja Department of Crop Physiology, University of Agricultural

Beata Orman Department of Genetics, Silesian University, Katowice, Poland

Ana Clara Pontaroli Department of Genetics, University of Georgia, Athens, GA,

T.G. Prasad Department of Crop Physiology, University of Agricultural Sciences,

Harsh Raman NSW Department of Industry and Investment, Wagga Wagga

Silvio Salvi Department of Agroenvironmental Sciences and Technology, Viale

Maria Corinna Sanguineti Department of Agroenvironmental Sciences and

M.S. Sheshshayee Department of Crop Physiology, University of Agricultural

Rogerio O. Sousa Department of soils, Eliseu Maciel School of Agronomy,

Rohini Sreevathsa Department of Crop Physiology, University of Agricultural

Namita Srivastava Department of Crop Physiology, University of Agricultural

Iwona Szarejko Department of Genetics, Silesian University, Katowice, Poland

Michael J. Thomson International Rice Research Institute (IRRI), DAPO 7777,

Roberto Tuberosa Department of Agroenvironmental Sciences and Technology,

M. Udayakumar Department of Crop Physiology, University of Agricultural

Giel E. van Noorden Australian Research Council Centre of Excellence for

Rajeev K. Varshney Centre of Excellence in Genomics (CEG), International

Ons Zahaf Institut des Sciences du Végétal, C.N.R.S, 91198 Gif-sur-Yvette,

Antonio Costa de Oliveira and Rajeev K. Varshney